EP1863926A2 - Compositions and methods for treating inflammatory cns disorders - Google Patents

Compositions and methods for treating inflammatory cns disorders

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Publication number
EP1863926A2
EP1863926A2 EP06725044A EP06725044A EP1863926A2 EP 1863926 A2 EP1863926 A2 EP 1863926A2 EP 06725044 A EP06725044 A EP 06725044A EP 06725044 A EP06725044 A EP 06725044A EP 1863926 A2 EP1863926 A2 EP 1863926A2
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EP
European Patent Office
Prior art keywords
park2
auts2
psenl
kcnip4
gene
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EP06725044A
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German (de)
French (fr)
Inventor
Daniel Cohen
Ilya Chumakov
Fabio Macciardi
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Ares Trading SA
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Ares Trading SA
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Priority to EP06725044A priority Critical patent/EP1863926A2/en
Publication of EP1863926A2 publication Critical patent/EP1863926A2/en
Withdrawn legal-status Critical Current

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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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates, generally, to methods and compositions for detecting or treating inflammatory CNS disorders, such as multiple sclerosis (MS).
  • the present invention more particularly relates to the human AUTS2, PARK2, PSENl and KCNIP4 genes, which can be used for the diagnosis, prevention and treatment of multiple sclerosis and related disorders, as well as for the screening of therapeutically active drugs.
  • the invention further discloses specific polymorphisms or alleles of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that are related to inflammatory CNS disorders such as multiple sclerosis, as well as diagnostic tools and kits based on these markers.
  • the invention can be used in the diagnosis or detection of the presence, risk or predisposition to, as well as in the prevention and/or treatment of multiple sclerosis and related disorders.
  • MS Multiple sclerosis
  • MS may result in the accumulation of various neurological disabilities.
  • Clinical disability in MS is presumed to be a result of repeated inflammatory injury with subsequent loss of myelin and axons, leading to tissue atrophy.
  • MS is manifested in physical symptoms (relapses and disability progression), Central Nervous System (CNS) inflammation, brain atrophy and cognitive impairment. Presenting symptoms include focal sensory deficits, focal weakness, visual problems, imbalance and fatigue. Sexual impairment and sphincter dysfunction may occur. Approximately half of the patients with MS may experience cognitive impairment or depression.
  • CNS Central Nervous System
  • MS is now considered to be a multi-phasic disease and periods of clinical quiescence (remissions) occur between exacerbations. Remissions vary in length and may last several years but are infrequently permanent.
  • Four courses of the disease are individualized: relapsing-remitting (RR), secondary progressive (SP), primary progressive (PP) and progressive relapsing (PR) multiple sclerosis. More than 80% of patients with MS will initially display a RR course with clinical exacerbation of neurological symptoms, followed by a recovery that may or may not be complete ⁇ Lublin andReingold, Neurology, 1996, 46:907-911).
  • MS onset is defined by the occurrence of the first neurological symptoms of CNS dysfunction.
  • CSF cerebrospinal fluid
  • MRI magnetic resonance imaging
  • the International Panel on the Diagnosis of MS issued revised criteria facilitating the diagnosis of MS and including MRI together with clinical and para-clinical diagnostic methods ⁇ Me Donald et al, 2001, Ann. Neurol, 50:121-127).
  • Molecules currently used for the treatment of multiple sclerosis in its various forms may have side effects and may act only against the symptoms of the disease. Consequently, there is a strong need for new molecules without or with less associated side effects that are directed against novel targets. Therefore, there is a need to identify proteins involved in the disease, thereby providing new targets allowing new screenings for drugs, resulting in new drugs that are efficient in treatment of this serious inflammatory CNS disease and related disorders.
  • the present invention now discloses novel approaches to the diagnosis and treatment of multiple sclerosis and related disorders, as well as for the screening of therapeutically active drugs.
  • the invention more specifically demonstrates that alterations in the AUTS2,
  • PARK2, PSENl and/or KCNIP4 gene are associated with the development of multiple sclerosis.
  • PSENl and/or KCNIP4 in particular, represent novel targets for therapeutic intervention against MS and related pathologies.
  • a first aspect of this invention thus resides in the use of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against multiple sclerosis and related disorders.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis or related disorders, comprising determining the ability of a compound to bind a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, or a fragment thereof, particularly of an allele of said gene or polypeptide that is associated with multiple sclerosis or a related disorder, or a fragment thereof.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis or related disorders, comprising testing for modulation of the activity of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, or a fragment thereof, particularly of an allele of said gene or polypeptide that is associated with multiple sclerosis or a related disorder, or a fragment thereof.
  • Another aspect of this invention resides in a method of assessing the presence of or predisposition to multiple sclerosis or a related disorder in a subject, comprising determining (in vitro or ex vivo) the presence of an alteration (e.g., a susceptibility mutation or allele) in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • an alteration e.g., a susceptibility mutation or allele
  • a further aspect of this invention relates to the use of a modulator of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, preferably an agonist thereof, for the preparation of a medicament for treating or preventing multiple sclerosis or a related disorder in a subject, as well as to corresponding methods of treatment.
  • the invention more specifically encompasses methods of treating multiple sclerosis or related disorders in a subject through a modulation of AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide expression or activity, preferably through an activation or restoration thereof.
  • Such treatments use, for instance, a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide, a AUTS2, PARK2, PSENl and/or KCNIP4 DNA sequence (including antisense sequences, RNAi), antibodies against AUTS2, PARK2, PSENl and/or KCNIP4 polypeptides, ligands of AUTS2, PARK2, PSENl and/or KCNIP4 or drugs that modulate, preferably mimic or stimulate, AUTS2, PARK2, PSENl and/or KCNIP4 expression or activity.
  • the invention particularly relates to methods of treating individuals having disease-associated alleles of the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
  • the invention further relates to the screening of alteration(s) associated with multiple sclerosis or related disorders in the AUTS2, PARK2, PSENl and/or KCNIP4 gene locus in patients. Such screenings are useful for diagnosing the presence, risk or predisposition to multiple sclerosis and related disorders, and/or for assessing the efficacy of a treatment of such disorders.
  • a further aspect of this invention includes nucleic acid probes and primers that allow specific detection of susceptibility markers in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA through selective hybridization or amplification.
  • the invention also encompasses particular nucleic acids, vectors and recombinant cells, as well as kits or solid phase bound nucleic acids or proteins such as DNA or protein arrays or chips suitable for implementing the above detection, screening or treatment methods.
  • the invention also discloses and encompasses markers in the AUTS2, PARK2, PSENl and/or KCNIP4 nucleic acids and polypeptides that are associated with multiple sclerosis and related disorders. Examples of such markers are more particularly selected from the markers as listed in Tables 2 to 5.
  • the invention can be used in the diagnosis of predisposition to, detection, prevention and/or treatment of multiple sclerosis and related disorders in any mammalian subjects, particularly human patients.
  • the present invention stems from association studies conducted on different MS populations, using a number of random markers. The results of these studies show that the AUTS2, PARK2, PSENl and KCNIP4 genes are strongly associated with multiple sclerosis, and that new and validated (biallelic) markers located in said gene or corresponding RNAs are associated with multiple sclerosis and related disorders.
  • the present invention thus provides novel means and methods to identify compounds useful in the treatment of multiple sclerosis and related disorders.
  • the invention further provides novel approaches to the detection, diagnosis and monitoring of multiple sclerosis or related disorders in a subject, as well as for genotyping of inflammatory CNS patients, in particular patients having MS.
  • multiple sclerosis may be defined as in the DSM-IV classification (Diagnosis and Statistical Manual of Inflammatory CNS Disorders, Fourth Edition, American Psychiatric Association, Washington D.C., 1994).
  • inflammatory CNS disorder includes in particular demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases. Inflammatory CNS disorders other than MS are also called MS-related diseases herein.
  • MS demyelinating inflammatory CNS disorders
  • PML progressive multifocal leukoencephalopathy
  • ADAM acute disseminated encephalomyelitis
  • MS-related diseases are also called MS-related diseases herein.
  • AUT S2 designates the human AUTS2 gene, as well as variants, analogs and fragments thereof.
  • the nucleic and amino acid sequences of a AUTS2 gene or polypeptide are available in the literature (AUTS2 GENE; AUTS2 (KIAA0442) - Gene map locus 7ql l.2 - Size: 1259 amino acids; 138981 Da. Two isoforms identified in SwissProt entry "AUTS2 HUMAN").
  • the human AUTS2 gene has been suggested to be implicated in autism, but prior to the present invention has not been linked to inflammatory CNS disorder such as demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases.
  • inflammatory CNS disorder such as demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases.
  • AUTS2 is identical to the KIAA0442 gene identified by Ishikawa et al. (DNA Res. 4: 307-313, 1997).
  • the predicted 1,259-amino acid protein contains 2 proline-rich domains and regions that share homology with the dwarfin family consensus sequence and with topoisomerase. It has a PY motif, several putative phosphorylation sites and N-myristoylation sites, and 2 putative N- glycosylation sites.
  • the AUTS2 protein shares 93% amino acid identity with its murine homolog.
  • AUTS2 Northern blot analysis detected strong expression of 7.5- and 8.0-kb transcripts in fetal and adult brain.
  • AUTS2 is also strongly expressed in skeletal muscle and kidney, with lower levels in placenta, lung, and leukocytes. In fetal brain, AUTS2 is expressed in frontal, parietal, and temporal lobes, but not in the occipital lobe.
  • Sultana et al. identified several smaller splice variants of AUTS2. By genomic sequence analysis, Sultana et al. (2002) determined that the AUTS2 gene spans 1.2 Mb and contains 19 exons.
  • PARK2 The nucleic and amino acid sequences of a PARK2 gene or polypeptide are available in the literature (PARKIN; PARK2 (PRKN - FRAGILE SITE FRA6E, INCLUDED) - Gene map locus 6q25.2-q27 - Size: 465 amino acids; 51650 Da - Localisation: cytoplasmic). 5 iso forms have been identified in SwissProt entry PRKN2 HUMAN.
  • the human PARK2 gene has been linked to Parkinson disease, but not inflammatory CNS disorder such as demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases.
  • CNS disorder such as demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases.
  • MS progressive multifocal leukoencephalopathy
  • ADAM acute disseminated encephalomyelitis
  • PARK2 forms an E3 ubiquitin ligase complex with UBE2L3 or UBE2L6 (part of a SCF- like complex, consisting of PARK2, CULl and FBXW7).
  • PARK2 interacts with SNCAIP and binds to the C2A and C2B domains of SYTl 1. It interacts and regulates the turnover of SEPT5. It is also part of a complex, including STUBl, HSP70 and GPR37. The amount of STUBl in the complex increases during ER stress. STUBl promotes the dissociation of HSP70 from PARK2 and GPR37, thus facilitating PARK2-mediated GPR37 ubiquitination.
  • HSP70 transiently associates with unfolded GPR37 and inhibits the E3 activity of PARK2, whereas, STUBl enhances the E3 activity of PARK2 through promotion of dissociation of HSP70 from PARK2-GPR37 complexes.
  • PARK2 interacts with PSMD4.
  • PARK2 is cytoplasmic and co-localizes with STYIl in neutrites.
  • PARK2 co-localizes as well with SNCAIP in brainstem Lewy bodies.
  • PARK2 auto-ubiquitinates in an E2-dependent manner leading to its own degradation.
  • PARK2 is S-nitrosylated.
  • the inhibition of PARK2 ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in PD by impairing the ubiquitination of PARK2 substrates.
  • the parkin locus (PRKN), adjacent to the 6q telomere is hyper-recombinable and lies within FRA6E, the third most common fragile site in tumor tissue.
  • PD parkinson's disease
  • PD is a complex, multifactorial disorder that typically manifests after the age of 50 years, although early- onset cases (before 50 years) are known.
  • PD is characterized by bradykinesia, resting tremor, muscular rigidity and postural instability, as well as by a clinically significant response to treatment with levodopa.
  • the pathology of PD involves the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (intraneuronal accumulations of aggregated proteins), in surviving neurons in various areas of the brain.
  • AR-jp autosomal recessive juvenile parkinsonism
  • EPDF early- onset parkinsonism with diurnal fluctuation
  • PDJ autosomal recessive juvenile Parkinson's disease
  • PARK2 Parkinson's disease type 2
  • AR-JP is symptomatically different in several aspects from idiopathic Parkinson's disease, although classic symptoms such as bradykinesia, rigidity and tremor are present. Additional clinical features include early DOPA-induced dyskinesia, diurnal fluctuation of the symptoms, sleep benefit, dystonia and hyper-reflexia.
  • AR-JP is usually characterized by onset before 40, with a mean age at onset of 23.2 years.
  • AR-JP patients show loss of dopaminergic neurons in the substantia nigra, similar to that seen in Parkinson's disease; however, Lewy bodies (intraneuronal accumulations of aggregated proteins) are absent. Defects in PARK2 may be involved in the development and/or progression of ovarian cancer.
  • PSENl designates the human PSENl gene, as well as variants, analogs and fragments thereof.
  • the nucleic and amino acid sequences of a PSENl gene or polypeptide are available in the literature.
  • the PSENl gene (PRESENILIN 1; PSENl (PSl, S182) - Gene map locus 14q24.3 - Size: 467 amino acids; 52667 Da) has previously been related to Alzheimers disease, but not to inflammatory CNS disorders, such as MS. 6 isoforms have been identified (see SwissProt etnry PSNl HUMAN). Heterogeneous proteolytic processing generates N-terminal and C- terminal fragments of approximately 35 and 20 kDa, respectively. PSENl is phosphorylated on serine residues.
  • PSENl is a probable catalytic subunit of the gamma-secretase complex, an endoprotease complex that catalyzes the intramembrane cleavage of integral membrane proteins such as Notch receptors and APP (beta-amyloid precursor protein). It requires the other members of the gamma-secretase complex to have a protease activity. It may play a role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane. PSENl is also believed to regulate epithelial- cadherin function.
  • KCNIP4 designates the human KCNIP4 gene, as well as variants, analogs and fragments thereof.
  • the nucleic and amino acid sequences of a KCNIP4 gene or polypeptide are available in the literature (POTASSIUM CHANNEL-INTERACTING PROTEIN 4 (KCNIP4; KCHIP4, CALSENILIN-LDCE PROTEIN; CALP) - Gene map locus 4 ⁇ l5.3).
  • 4 alternative transcripts of KCNIP4 have been identified (REFSEQ proteins NP_079497.2, NP_671710.1, NP_671711.1, NP_671712.1).
  • Heterogeneous proteolytic processing generates N-terminal and C-terminal fragments of approximately 35 and 20 kDa, respectively.
  • KCNIP4 is phosphorylated on serine residues.
  • K channel-interacting proteins such as KCNIP4, specifically modulate the activity of Kv4 A-type potassium channels (KCNDl).
  • Kv4 A-type channels are known to contribute to the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart (Holmqvist et al., Proc. Nat. Acad. Sci. 99: 1035-1040, 2002).
  • KCNIP4 has not been linked to inflammatory CNS disorders such as for example MS.
  • Kchip4 Holmqvist et al. (2002) cloned mouse Kcnip4, which they designated Kchip4.
  • KCNIP4 splice variants that differ from each other mostly in the N-terminal domain.
  • Human and mouse variants that include an N-terminal 34-amino acid K channel inactivation suppressor (KIS) domain share 100% amino acid identity.
  • Northern blot analysis of rat tissues detected Kcnip4 expression in brain, but not in heart or any other tissue examined.
  • RT-PCR of mouse tissues detected expression of 2 splice variants in brain, but not in atrium or ventricle.
  • KCNIP4 By yeast 2-hybrid analysis, Morohashi et al. (Biol. Chem. 277: 14965-14975, 2002) determined that KCNIP4 interacts with the C termini of PS2 and PSl. By Ca(2+) overlay assay, they determined that the EF hand motif of KCNIP4 binds calcium. Using mutation analysis and coimmunoprecipitation experiments, they showed that the EF hand motif mediates the interaction between KCNIP4 and rat Kv4. Overexpression of KCNIP4 did not alter the metabolism or stability of PS, but overexpression of KCNIP4 with rat Kv4.2 (KCND2;) reconstituted the features of A-type K+ currents. Morohashi et al. (2002) concluded that KCNIP4 is a novel EF hand protein that interacts with both PS and Kv4.
  • gene shall be construed to include any type of coding nucleic acid region, including genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semisynthetic DNA, any form of corresponding RNA (e.g., mRNA), etc., as well as non coding sequences, such as introns, 5'- or 3 '-untranslated sequences or regulatory sequences (e.g., promoter or enhancer), etc.
  • the term gene particularly includes recombinant nucleic acids, i.e., any non naturally occurring nucleic acid molecule created artificially, e.g., by assembling, cutting, ligating or amplifying sequences.
  • a gene is typically double-stranded, although other forms may be contemplated, such as single-stranded. Genes may be obtained from various sources and according to various techniques known in the art, such as by screening DNA libraries or by amplification from various natural sources. Recombinant nucleic acids may be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. The term “gene” may comprise any and all splicing variants of said gene.
  • a fragment of a gene designates any portion of at least about 8 consecutive nucleotides of a sequence of said gene, preferably at least about 15, more preferably at least about 25 nucleotides, further preferably of at least 35, 50, 75, 100, 150, 200 or 300 nucleotides. Fragments include more particularly all possible nucleotide length between 8 and 500 nucleotides, preferably between 15 and 300, more preferably between 25 and 200.
  • a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide designates any protein or polypeptide encoded by a AUTS2, PARK2, PSENl and/or KCNIP4 gene as disclosed above, respectively.
  • polypeptide designates, within the context of this invention, a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
  • a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide also denotes a polypeptide, which is specific fragment of AUTS2, PARK2, PSENl and/or KCNIP4 of at least 8, 15, 20, 50, 100, 250, 300 or 350 amino acids in length.
  • This term also does not specify or exclude post-translational or post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
  • polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • amino acid including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.
  • polypeptides with substituted linkages as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • Fusion proteins are useful for generating antibodies against a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide and for use in various assay systems.
  • fusion proteins can be used to identify proteins, which interact with portions of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
  • Protein affinity chromatography or library- based assays for protein-protein interactions such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises at least 25, 50, 75, 100, 150, 200, 300, 350 or 372 contiguous amino acids of SEQ ID NO: 2.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include beta- galactosidase, beta-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a fusion protein also can be engineered to contain a cleavage site located between the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide-encoding sequence and the heterologous protein sequence, so that the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences for AUTS2, PARK2, PSENl and/or KCNIP4 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • treat or “treating” as used herein is meant to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.
  • treatment as used herein also encompasses the term “prevention of the disorder”, which is, e.g., manifested by delaying the onset of the symptoms of the disorder to a medically significant extent. Treatment of the disorder is, e.g., manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.
  • modulated or modulation or regulated or “regulation” as used herein refer to both upregulation [i.e., activation or stimulation (e.g., by agonizing or potentiating)] and downregulation [i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)].
  • oligonucleotides and “polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • nucleotide as used herein as an adjective to describe compounds comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a compound, or individual unit in a larger nucleic acid compound, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
  • nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO95/04064, the disclosure of which is incorporated herein by reference.
  • the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides.
  • the polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
  • primer denotes a specific oligonucleotide sequence, which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
  • a primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.
  • Typical primers of this invention are single- stranded nucleic acid molecules of about 6 to 50 nucleotides in length, more preferably of about 8 to about 40 nucleotides in length, typically of about 16 to 25.
  • the Tm is typically of about 60°C or more.
  • the sequence of the primer can be derived directly from the sequence of the target gene. Perfect complementarity between the primer sequence and the target gene is preferred, to ensure high specificity. However, certain mismatch may be tolerated.
  • probe denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.
  • Probes of this invention typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 750, more preferably of between 15 and 600, typically of between 20 and 400.
  • the sequence of the probes can be derived from the sequences of the AUTS2, PARK2, PSENl and/or KCNIP4 gene sequence.
  • the probe may contain nucleotide substitutions and/or chemical modifications, e.g., to increase the stability of hybrids or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, etc.
  • complementary or “complement thereof are used herein to refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
  • non-human animal refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice.
  • animal is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term "non-human”.
  • twin and phenotype are used interchangeably herein and refer to any clinically distinguishable, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example.
  • phenotype are used herein to refer to symptoms of, or susceptibility to inflammatory CNS disorder; or to refer to an individual's response to an agent acting on inflammatory CNS disorder; or to refer to symptoms of, or susceptibility to side effects to an agent acting on inflammatory CNS disorder.
  • allele refers to one of the variant forms of a biallelic or multiallelic marker, differing from other forms in its nucleotide sequence. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
  • polymorphism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. "Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population.
  • a "polymorphic site” is the locus at which the variation occurs.
  • a polymorphism may comprise a substitution, deletion or insertion of one or more nucleotides.
  • a single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site.
  • SNP single nucleotide polymorphism
  • the present invention provides novel means and methodologies for detecting or diagnosing multiple sclerosis and related disorders in a human subject.
  • the present methods may be implemented at various development stages of said pathologies, including early, pre- symptomatic stages, and late stages, in adults, children and pre-birth.
  • the invention is suited to determine the prognosis, to assess a predisposition to or a risk of development of pathology, to characterize the status of a disease or to define the most appropriate treatment regimen for a patient.
  • a particular object of this invention resides in a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence of an alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • Another object of this invention relates to methods of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder, the methods comprising detecting the presence of an alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
  • Medications for MS within the meaning of the present invention include the four FDA approved immunomodulating agents for RRMS: three beta interferons (Betaseron®, Berlex; Avonex®, Biogen; Rebif®, Serono) and Glatimarer Acetate (Copaxone®, Amgen). Medications for MS within the meaning of the present invention also include the FDA approved immunosuppressing drug for worsening MS, Mitoxantrone (Novantrone®, Amgen).
  • interferon and "interferon-beta (IFN-beta)", as used herein, are intended to include fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments.
  • IFN-beta suitable in accordance with the present invention is commercially available e.g. as Rebif® (Serono), Avonex® (Biogen) or Betaferon® (Schering).
  • Rebif® Sterotono
  • Avonex® Biogen
  • Betaferon® Schering
  • the use of interferons of human origin is also preferred in accordance with the present invention.
  • the term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof.
  • Rebif® (recombinant human interferon-) is the latest development in interferon therapy for multiple sclerosis (MS) and represents a significant advance in treatment.
  • Rebif® is interferon (IFN)-beta Ia, produced from mammalian cell lines. It was established that interferon beta- Ia given subcutaneously three times per week is efficacious in the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS).
  • Interferon beta- Ia can have a positive effect on the long-term course of MS by reducing number and severity of relapses and reducing the burden of the disease and disease activity as measured by MRI.
  • the dosing of IFN- ⁇ in the treatment of relapsing-remitting MS according to the invention depends on the type of IFN- ⁇ used.
  • IFN is recombinant IFN- ⁇ Ib produced in E. CoIi, commercially available under the trademark Betaseron
  • IFN may preferably be administered sub-cutaneously every second day at a dosage of about of 250 to 300 g or 8 MIU to 9.6 MIU per person.
  • IFN is recombinant IFN- ⁇ Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Avonex
  • IFN may preferably be administered intra-muscularly once a week at a dosage of about of 30g to 33 g or 6 MIU to 6.6 MIU per person.
  • IFN when IFN is recombinant IFN- ⁇ Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Rebif, it may preferably be administered sub-cutaneously three times a week (TIW) at a dosage of 22 to 44 g or 6 MIU to 12 MIU per person.
  • TIW sub-cutaneously three times a week
  • the alteration ("susceptibility alteration") in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide may be any susceptibility marker in said gene or polypeptide, i.e., any nucleotide or amino acid alteration associated to multiple sclerosis or a related disease.
  • An alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertion(s) in the coding and/or non- coding region of the gene, either isolated or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene. Typical deletions affect small regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene.
  • Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene. Rearrangements include for instance sequence inversions.
  • An alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene may also be an aberrant modification of the polynucleotide sequence, such as of the methylation pattern of the genomic DNA, allelic loss of the gene or allelic gain of the gene.
  • the alteration may be silent (i.e., create no modification in the amino acid sequence of the protein), or may result, for instance, in amino acid substitutions, frameshift mutations, stop codons, RNA splicing, e.g.
  • RNA or protein instability or a non-wild type level of the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
  • alteration may result in the production of a polypeptide with altered function or stability, or cause a reduction or increase in protein expression levels. Typical alterations are single nucleotide substitutions.
  • the chromosomal SNP positions are based on NCBI human genome Build 35. These position show associated alleles which are preferred markers of the invention.
  • the sites of the SNPs ("site") are further identified by unique Affymetrix SNP identifiers referring to the Affymetrix genechip human mapping IOOK set. These Affymetrix identifiers refer to unique oligonucleotides of 25bp length which allow for an identification of the respective SNP independent of the sequence of a specific genome build/version.
  • the present invention now discloses several markers or mutations in the AUTS2 gene, which are associated with multiple sclerosis. These mutations are reported in tables 2a and 2b. Preferred genetic alterations are disclosed in table 2a below. Most preferred alterations are disclosed in table 2b below.
  • association results of the single biallelic marker frequency analysis show that the AUTS2 gene is associated with multiple sclerosis.
  • a preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 2a in the AUT S2 gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 2b, or any combination thereof.
  • a preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 2a in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • one or more markers, as well as any combination thereof are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the additional presence of an associated allele in the PARK2, PSENl and/or KCNIP4 gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the present invention now discloses several markers or mutations in the PARK2 gene, which are associated with multiple sclerosis. These mutations are reported in table 3 (table 3a and 3b). Preferred genetic alterations are disclosed in table 3a below. Most preferred alterations are disclosed in table 3b below.
  • association results of the single biallelic marker frequency analysis show that the PARK2 gene is associated with multiple sclerosis.
  • a preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 3a in the PARK2 gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 3b, or any combination thereof.
  • a preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 3a or 3b in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • one or more markers are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the additional presence of an associated allele in the AUTS2, PSENl and/or KCNIP4 gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the present invention now discloses several markers or mutations in the PSENl gene, which are associated with multiple sclerosis. These mutations are reported in table 4 (table 4a and table 4b). Preferred genetic alterations are disclosed in table 4a below. Most preferred alterations are disclosed in table 4b below.
  • association results of the single biallelic marker frequency analysis show that the PSENl gene is associated with multiple sclerosis.
  • a preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 4a in the PSENl gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 4b, or any combination thereof.
  • a preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 4a or 4b in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • one or more markers are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the additional presence of an associated allele in the AUTS2, PARK2, and/or KCNIP4 gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the present invention now discloses several markers or mutations in the KCNIP4 gene, which are associated with multiple sclerosis. These mutations are reported in table 5 (tables 5a and 5b). Preferred genetic alterations are disclosed in table 5a below. Most preferred alterations are disclosed in table 5b below.
  • association results of the single biallelic marker frequency analysis show that the KCNIP4 gene is associated with multiple sclerosis.
  • a preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 5a in the KCNIP4 gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 5b, or any combination thereof.
  • a preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 5a or 5b in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • one or more markers are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the additional presence of an associated allele in the AUTS2, PARK2, and/or PSENl gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the presence of an alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene may be detected by any technique known per se to the skilled artisan (reviewed by Kwok et al., 2003), including sequencing, pyrosequencing, selective hybridisation, selective amplification and/or mass spectrometry including matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) (Gut et al., 2004).
  • the alteration is detected by selective nucleic acid amplification using one or several specific primers.
  • the alteration is detected by selective hybridization using one or several specific probes.
  • Further techniques include gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing; fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping; rolling circle amplification and Invader assays as well as DNA chip-based microarray and mass spectrometry genotyping technologies (Shi et al., 2001).
  • gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing
  • fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homo
  • RNA expression of altered genes can be quantified by methods known in the art such as subtractive hybridisation, quantitative PCR, TaqMan, differential display reverse transcription PCR, serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips.
  • Particular methods include allele-specific oligonucleotide (ASO), allele-specific amplification, fluorescent in situ hybridization (FISH) Southern and Northern blot, and clamped denaturing gel electrophoresis.
  • Protein expression analysis methods are known in the art and include 2-dimensional gel- electrophoresis, mass spectrometry and antibody microarrays (Freeman et al., 2004 and Zhu et al., 2003).
  • Sequencing can be carried out using techniques well known in the art, using automatic sequencers.
  • the sequencing may be performed on the complete gene or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.
  • Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA). These techniques can be performed using commercially available reagents and protocols.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • a preferred technique is allele-specific PCR.
  • Nucleic acid primers useful for amplifying sequences from the AUTS2, PARK2, PSENl and/or KCNIP4 gene are able to specifically hybridize with a portion of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that either flanks or overlaps with an alteration, such as a susceptibility marker.
  • the primer sequence overlaps with the alteration when said alteration is contained within the sequence of the AUTS2, PARK2, PSENl and/or KCNIP4 gene to which the primer hybridizes.
  • the primer sequence flanks the alteration when the primer hybridizes with a portion of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that is preferably located at a distance below 300 bp of said alteration, even more preferably below 250, 200, 150, 100, 50, 40, 30 or 20 bp from said alteration.
  • the primer hybridizes with a portion of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that is at 5, 4, 3, 2, 1 bp distance or immediately adjacent to said alteration.
  • the invention also relates to 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 multiple sclerosis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder.
  • the methods involve the use of a nucleic acid probe specific for a AUTS2, PARK2, PSENl and/or KCNIP4 or altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA, followed by the detection of the presence of a hybrid.
  • the probe may be used in suspension or immobilized on a substrate or support.
  • the probe is typically labelled to facilitate detection of hybrids.
  • a specific object of this invention is a nucleic acid probe complementary to and specific for a region of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA that carries an alteration as described in Table 2a to 5a and preferably Table 2b to 5b.
  • the probes of the present invention are, more preferably, capable of discriminating between an altered and non-altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA sequence, i.e., they specifically hybridise to a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA carrying a particular alteration as described above, and essentially do not hybridise under the same hybridization conditions or with the same stability to a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA lacking said alteration.
  • 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 multiple sclerosis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder.
  • the detection methods can be performed in vitro, ex vivo or in vivo, preferably in vitro or ex vivo. They are typically performed on a sample from the subject, such as any biological sample containing nucleic acids or polypeptides. Examples of such samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma, saliva, urine, seminal fluid, etc.
  • the sample may be collected according to conventional techniques and used directly for diagnosis or stored. In particular, they may be obtained by non-invasive methods, such as from tissue collections.
  • the sample may be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing.
  • Treatments include, for instant, lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, etc.
  • the nucleic acids and/or polypeptides may be pre- purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. Considering the high sensitivity of the claimed methods, very few amounts of sample are sufficient to perform the assay.
  • the sample is typically contacted with probes or primers as disclosed above.
  • Such contacting may be performed in any suitable device, such as a plate, tube, well, glass, etc.
  • the contacting may be performed on a substrate coated with said specific reagents, such as a nucleic acid array.
  • the substrate may be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids of the sample.
  • the finding of an altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA or polypeptide in the sample is indicative of the presence, predisposition or stage of progression of multiple sclerosis or a related disorder in the subject.
  • one only of the above-disclosed markers is assessed, or several of them, in combination(s).
  • a method of treating multiple sclerosis or a related disorder in a subject in need of such treatment comprising of administering to the subject a compound that modulates the synthesis, expression or activity of one or more of the genes or gene products of the genes listed in Table 2a to 5a and preferably Table 2b to 5b in a therapeutically effective amount so that at least one symptom of the multiple sclerosis or a related disorder is ameliorated.
  • a method of treating multiple sclerosis or a related disorder in a subject in need of such treatment wherein the subject has a susceptibility alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene comprising of administering to the subject a therapeutically effective amount of a medication for MS, such as for example interferon-beta, preferably interferon-beta Ia.
  • the susceptibility alteration is selected from one or more of the susceptibility markers listed in Table 2a to 5a and preferably Table 2b to 5b.
  • the susceptibility alteration is located within the 3' or 5' region of the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
  • the susceptibility susceptibility alteration is a single nucleotide mutation.
  • kits for the identification of a genetic polymorphism pattern at the AUTS2, PARK2, PSENl and/or KCNIP4 gene associated with increased risk of the presence of or predisposition to multiple sclerosis or a related disorder in a subject comprising:
  • (b) means for determining a genetic polymorphism pattern for the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
  • the present invention also provides novel targets and methods for the screening of drug candidates or leads.
  • These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
  • a particular object of this invention resides in the use of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide as a target for screening candidate drugs for treating or preventing multiple sclerosis or a related disorder.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide with a candidate compound, and selecting compounds that bind said AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide at the surface of said cells and/or that modulate the activity of the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a neuroactive compound, and further preferably a compound that can be used for treating multiple sclerosis or a related disorder, or as a lead to develop drugs for treating multiple sclerosis or a related disorder.
  • a “biologically active” compound preferably is a compound that modulates the activity of AUTS2, PARK2, PSENl and/or KCNIP4.
  • the above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of multiple sclerosis or a related disorder, such as an animal model.
  • Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to multiple sclerosis or a related disorder in a subject.
  • the determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc.
  • the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
  • Modulation of activity includes, without limitation, stimulation of the surface expression of the AUTS2, PARK2, PSENl and/or KCNIP4 receptor, modulation of multimerization of said receptor (e.g., the formation of multimeric complexes with other sub-units), etc.
  • the cells used in the assays may be any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide) or any cell that expresses an endogenous AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
  • Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • prokaryotic cells such as bacteria
  • eukaryotic cells such as yeast cells, mammalian cells, insect cells, plant cells, etc.
  • yeast cells such as yeast cells, mammalian cells, insect cells, plant cells, etc.
  • E.coli E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts
  • mammalian cell lines e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • Preferred selected compounds are agonists of AUTS2, PARK2, PSENl and/or KCNIP4, i.e., compounds that can bind to AUTS2, PARK2, PSENl and/or KCNIP4 and mimic the activity of an endogenous ligand thereof.
  • the screening assays of the present invention use, either alone or in addition to another AUTS2, PARK2, PSENl and/or KCNIP4 sequence, an altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, particularly a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide having a mutation as listed in Table 2a to 5a and preferably Table 2b to 5b, respectively.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a AUTS2, PARK2, PSENl and/or KCNIP4 gene according to the present invention and determining the ability of said test compound to modulate the expression of said AUTS2, PARK2, PSENl and/or KCNIP4 gene, preferably to stimulate expression thereof.
  • this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a AUTS2, PARK2, PSENl and/or KCNIP4 gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
  • this invention relates to the use of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide or fragment thereof, whereby the fragment is preferably a AUTS2, PARK2, PSENl and/or KCNIP4 gene-specific fragment, for isolating or generating an agonist or stimulator of the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide for the treatment of multiple sclerosis or a related disorder, wherein said agonist or stimulator is selected from the group consisting of:
  • a specific antibody or fragment thereof including a) a chimeric, b) a humanized or c) a fully human antibody as well as
  • an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
  • the present invention now discloses novel approaches to the treatment of multiple sclerosis and related disorders by modulating the activity or expression of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide. More particularly, the present invention provides the first evidence of a correlation between said gene and said diseases in human subjects, and allows the design of novel therapeutic approaches based on a modulation, preferably a stimulation or increase of a AUTS2, PARK2, PSENl and/or KCNIP4 activity.
  • a particular object of this invention resides in the use of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide, or a nucleic acid encoding the same, for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
  • a further object of this invention resides in the use of a modulator of AUTS2, PARK2, PSENl and/or KCNIP4 for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
  • the modulator is an agonist or activator of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
  • the agonist is a natural ligand of AUTS2, PARK2, PSENl and/or KCNIP4, or an antibody, such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non-peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), that selectively binds AUTS2, PARK2, PSENl and/or KCNIP4.
  • an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non-peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e
  • the modulator is an inhibitor or antagonist of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
  • a further object of this invention resides in a pharmaceutical composition comprising a nucleic acid encoding a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide or a vector encoding the same, and a pharmaceutically acceptable carrier or vehicle.
  • compositions are particularly suited for treating or preventing multiple sclerosis or a related disorder in a subject presenting an alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, particularly in a subject presenting a marker as described in Table 2a to 5a and preferably Table 2b to 5b, respectively.
  • Another object of this invention is an isolated or recombinant AUTS2, PARK2, PSENl and/or KCNIP4 gene or a fragment thereof, wherein said gene or fragment comprises a marker selected Table 2a to 5a and preferably Table 2b to 5b, respectively.
  • the invention also relates to any vector comprising a nucleic acid as defined above.
  • the vector may be any plasmid, phage, virus, episome, artificial chromosome, and the like.
  • the vector is a recombinant virus.
  • Viral vectors may be produced from different types of viruses, including without limitation baculoviruses, retroviruses, adenoviruses, AAVs, etc., according to recombinant DNA techniques known in the art.
  • the recombinant virus is typically replication-defective, even more preferably selected from El- and/or E4-defective adenoviruses, Gag-, pol- and/or env-defective retroviruses and Rep- and/or Cap-defective AAVs.
  • Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in
  • a further aspect of this invention is a recombinant host cell comprising a vector or a nucleic acid as defined above.
  • the recombinant cell may be any prokaryotic or eukaryotic cells as discussed above.
  • the recombinant cell preferably expresses a recombinant AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide at its surface.
  • a preferred embodiment of the invention is the use of an activator or agonist of AUTS2, PARK2, PSENl and/or KCNIP4 or a receptor comprising AUTS2, PARK2, PSENl and/or KCNIP4 in the preparation of a medicament for the treatment of multiple sclerosis or a related disorder.
  • a particularly preferred embodiment of the invention is the use of an activator or agonist of AUTS2, PARK2, PSENl and/or KCNIP4 or a receptor comprising AUTS2, PARK2, PSENl and/or KCNIP4 in the preparation of a medicament for the treatment of multiple sclerosis or a related disorder
  • the activator or agonist is an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • the invention also relates to a method of treating or preventing multiple sclerosis or a related disorder in a subject, the method comprising administering to said subject a compound that modulates, preferably that activates or mimics, expression or activity of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide as defined above.
  • a particular embodiment of the present invention resides in a method of treating or preventing multiple sclerosis or a related disorder in a subject, the method comprising (i) detecting in a sample from the subject the presence of an alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide as defined above and (ii) administering to said subject an agonist of AUTS2, PARK2, PSENl and/or KCNIP4.
  • said alteration is selected from the group consisting of an alteration as disclosed in Table 2a to 5a and preferably Table 2b to 5b, respectively.
  • the study comprised three collections of unrelated patients with multiple sclerosis (MS) and unrelated healthy controls recruited from the neurological Department of Rennes (France: 314 cases; 353 controls), Huddinge (Sweden: 279 cases; 301 controls) hospitals and SeraCare (USA: 289 cases; 289 controls).
  • Table 1 provides a summary for the description and stratification study of the different collections.
  • RR relapsing-remitting
  • SP relapsing-secondary progressive
  • PP primary-progressive
  • EDSS Kurtzke Expanded Disability Status Scale
  • Rennes collection Each patient and control subject included in the analysis had to be born in Bretagne, France as well as their parents and grand-parents.
  • the female / male ratio in the patient group was 2.14 (214 Females & 100 Males) with a mean age of 44 [19;68] years and in the control group 2.07 (241 Females & 116 Males) with a mean age of 35 [18;56] years.
  • the female / male ratio in the patient group was 2.4 (196 Females & 83 Males) with a mean age of 47 [22;75].
  • the control group in Huddinge collection included 301 (214 Females & 87 Males) healthy volunteers and the Female / male ratio was 2.5. Ages ranged from 22 to 73 years with a mean age of 47 years.
  • the group of cases included 289 subjects with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 50 [32;74] years.
  • the group of healthy volunteers included 289 individuals with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 48.7 [36;75] years.
  • Genomic DNA was extracted from EDTA anticoagulated peripheral blood according to a Standard proteinase K digestion and a modified salting out extraction method of Miller and co-workers (1988).
  • Each group, or panel of 12 markers must be of the same extension type for processing on the UHT since each extension mix contains two labeled terminators (Bodipy-Fluorescein and TAMRA). Each group of twelve is referred to as a panel of markers.
  • Autoprimer.com automatically optimizes the grouping of the markers by extension mix and appends tag sequences to the 5' ends of the SNP-IT primers, which are complementary to the tags immobilized on the microarray plate.
  • a five-micro liter PCR was performed in 384-well plates (MJ Research, Watertown, MA, USA) using 75-uM dNTPs and 0.5U AmpliTaq® Gold (Applied Biosystems) in IXPCR buffer. Two nanograms of genomic DNA were used in each reaction.
  • the 24 PCR primers were pooled and added such that each was at a final concentration of 50 nM.
  • Thermal cycling was performed in DNA Engine Tetrad thermal cyclers (MJ Research) using the following program: 95°C for 5 seconds followed by 45 cycles of 95°C for 30 seconds; 50°-55°C for 55 seconds; 72°C for 30 seconds. The first six cycles used an annealing temperature of 50°C after which the annealing temperature was increased by 0.2°C in the subsequent cycles until the annealing temperature reached 55°C. After the last cycle, the reaction was held at 72°C for 7 minutes followed by a 4°C hold.
  • hybridization buffer 5M NaCl, 0.5 M EDTA, 580 mM morpholinoethane sulphonic acid (MES) pH 6.6, IX Denhardt's Solution
  • MES morpholinoethane sulphonic acid
  • the SNPstream Array Imager is based upon a two-laser, two-color approach. Each sample is illuminated with a 488-nm laser beam and subsequently with a 532-nm laser beam to excite the fluorescent oligonucleotides captured on the UHT microarray plates.
  • the system 5 contains two emission band filters. Fluorescence emission from 488-nm excitation (Bodipy- Fluorescein) is captured in a band 50 nm wide, centered at 535nm. Fluorescence emission from 532-nm excitation (TAMRA) is captured in a band 55 nm wide, centered at 590 nm.
  • a colorcorrected custom lens, of high numerical aperture and 100-um A 2 X3 well area is imaged per frame. Sixty- four 2 X3 well images/color are taken per plate for a 10 total of 384 wells. Total time required for the process is approximately seven minutes/plate.
  • Genome Calls from spots detected using the SNPstream UHT Array Imager involves two discrete steps. First, the location and intensity of a spot within the well and 15 plate is determined for each wavelength; second, a genotype call is made based on the relative fluorescent intensities of each spot. Once a genotype call has been made, results are written to an Oracle® database where the data can easily be retrieved for viewing.
  • Spot detection is an automatic process performed by UHTImage software. Positive controls in each well are used to align the grids around the 4 X4 element array. Once a grid 20 is drawn, each spot is analyzed for morphology (i.e., circular shape and regular pixel intensity across each spot). Spots with low intensity or unusual morphology are marked as empty or fail. For each spot that passes the morphology test, an intensity value is generated and loaded into the UHT database. Failed spots are carried through the analysis but are flagged for the user to review.
  • morphology i.e., circular shape and regular pixel intensity across each spot.
  • Genotype calling is performed once all spot intensities are in the database for each sample within a plate.
  • Each SNP marker is analyzed separately using UHT GetGenossoftware. This software automatically creates genotype calls based on the intensity value of each spot at each wavelength for a given sample. These calls are based on how the sample points cluster when plotted on a X, Y graph where X corresponds to the intensity in the
  • UHT GetGenos uses a proprietary algorithm to determine the clusters and the genotypes for each sample. After the genotype calling, the results are stored in the database by microarray plate number, well, and spot location.
  • Affymetrix genotyping technology Use of Affymetrix genotyping technology have been described in Kennedy, G.C., et al. Nature Biotechnology 21, 1233-1237, 2003 (Large-scale genotyping of complex DNA). ; Liu, W.M., et al. Bioinformatics 19, 2397-2403, 2003. (Algorithms for Large Scale Genotyping Microarrays) ;Matsuzaki, H., et al. Genome Research 3, 414-25, 2004. (Parallel Genotyping of over 10,000 SNPs using a One Primer Assay on a High Density Oligonucleotide Array) ;Paez, J.G., et al. Nucleic Acids Research 32(9), 2004.
  • genomic DNA For each individual assayed, 250 ng of genomic DNA are digested separately with 10 U of Xbal or HmdIII (New England BioLabs) in volumes of 20 ⁇ L for 2 hours at 37 °C. Following heat inactivation at 70 °C for 20 minutes, 0.25 ⁇ M of Xbal adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -CTA GAG ATC AGG CGT CTG TCG TGC TCA TAA- 3') (Affymetrix), or HmdIII adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -AGC TAG ATC AGG CGT CTG TCG TGC TCA TAA-3') (Affymetrix) are ligated to the digested DNAs with T4 DNA Ligase (New England BioLabs) in 25 ⁇ L for 2 hours at 16 °C.
  • T4 DNA Ligase
  • PCR contains 10 ⁇ L of the diluted ligation reactions (25 ng of starting DNA) in 100 ⁇ L volumes containing 1.0 ⁇ M of primer (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3'), 0.30 mM dNTPs, 1.0 mM MgSO4, 5 U Platinum® Pfa Polymerase (Invitrogen), PCR Enhancer (Invitrogen) and Pfa Amplification Buffer (Invitrogen).
  • PCRs 30 cycles of PCRs are run with the following cycling program: 94 °C denaturation for 15 seconds, 60 °C annealing for 30 seconds, and 68 °C extension for 60 seconds.
  • 3 ⁇ L of PCR products are visualized on 2% TBE agarose gels to confirm the size range of amplicons.
  • the PCR products are purified over MinElute 96 UF PCR Purification plates (Qiagen), and recovered in 40 ⁇ L of EB buffer (Qiagen). PCR yields are measured by absorbance readings at 260 nm, and adjusted to a concentration of 40 ⁇ g per 45 ⁇ l.
  • the PCR products are fragmented to ⁇ 100 bp with DNAse I.
  • 0.20 U of DNAse I (Affymetrix) is added to 40 ug of purified PCR amplicons in a 55 ⁇ L volume containing Fragmentation Buffer (Affymetrix) for 35 minutes at 37 °C, followed by heat inactivation at 95 °C for 15 minutes. Fragmentation products are visualized on 4% TBE agarose gels.
  • the 3' ends of the fragmented amplicons are biotinlyated by adding 214 ⁇ M of a proprietary DNA labeling reagent (Affymetrix) using Terminal Deoxynucleotidyl Transferase (Affymetrix) in 70 ⁇ L volumes for 2 hours at 37 °C, followed by heat inactivation at 95 °C for 15 minutes.
  • Affymetrix a proprietary DNA labeling reagent
  • Affymetrix Terminal Deoxynucleotidyl Transferase
  • the fragmented and biotinylated PCR amplicons are combined with 11.5 ⁇ g/mL human Cot-1 (Invitrogen) and 115 ⁇ g/mL herring sperm (Promega) DNAs.
  • the DNAs are added to a hybridization solution containing 2.69 M tetramethylamonium chloride (TMACl), 5.77 mM EDTA, 56 mM MES, 5 % DMSO, 2.5 X Denhardt's solution, and 0.0115% Tween-20 in a final volume of 260 ⁇ L.
  • TMACl tetramethylamonium chloride
  • the hybridization solution was heated to 95 °C for 10 minutes then placed on ice.
  • Hybridizations are carried out at 48 °C for 16 to 18 hours in a rotisserie rotating at 60 rpm. Following the overnight hybridization, the arrays are washed with 6X SSPE and 0.01% Tween-20 at 25 °C, then more stringently washed with 0.6X SSPE and 0.01% Tween-20 at 45 °C.
  • Hybridization signals are generated in a three step signal amplification process: lO ⁇ g/mL streptavidin R-phycoerythrin (SAPE) conjugate (Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 °C; followed by the addition of 5 ⁇ g/mL biotinylated goat anti-streptavidin (Vector) to increase the effective number of biotin molecules on the target; and finally SAPE is added once again and washed extensively with 6X SSPE and 0.01% Tween-20 at 30 °C.
  • SAPE streptavidin R-phycoerythrin conjugate
  • the SAPE and antibody were added to arrays in 6X SSPE, IX Denhardt's solution and 0.01% Tween-20 at 25 °C for 10 minutes each. Following the final wash, the arrays are kept in Holding buffer (10OmM MES, IM [Na+], 0.01% Tween-20). The washing and staining procedures are run on Affymetrix fluidics stations. Arrays are scanned using GC S3000 scanners with AutoLoaders (Affymetrix). Scan images are processed to get hybridization signal intensity values using GCOS 2.0 software (Affymetrix). The DM genotype calling algorithm is implemented in GenoTyping Tools (GTT) (Affymetrix) and GDAS 3.0 (Affymetrix) analysis software.
  • GTT GenoTyping Tools
  • GDAS 3.0 Affymetrix
  • a stratification effect is a non-homogeneous representation of populations between the case and the control groups due to genetic heterogeneity, which may lead to spurious association results and replication problems.
  • cases and controls contain an admixture of different groups (for example, based on ethnicity), we expect to find a consistent pattern of allele-frequency differences between cases and controls, at many random loci throughout the genome, this difference exceeding the significant p-value for association at more than 5% of these random loci.
  • Fst test (Wright 1951) is an ANOVA-based method. The Fst value quantifies the loss of heterozygosity due to existence of a hierarchical structure. If it is different from 0, it means that the population under study are genetically heterogeneous, since allelic frequencies are different between populations. 2. Pritchard & Rosenberg test (Am. J.Hum. Genet. 65:220-228, 1999) calculates an overall chi-square statistic of allelic frequency differences between cases and controls.
  • Genomic Control ⁇ Devlin and Roeder 1999 Given that in the presence of population substructure, the standard chi-square statistic is inflated by a multiplicative factor, which is proportional to the degree of stratification, we can estimate and incorporate this multiplicative factor (lambda) into the disease - marker association tests (by rescaling the chi-square statistic) to correct for background population differences.
  • HWE Hardy- Weinberg law regulating equilibrium
  • control population used in case-control association studies must respect this equilibrium, if sampled randomly.
  • population of cases can present some disequilibrium that may point to "mutations" underlying the disease, since cases are not a random representation of the general population.
  • HWE test therefore serves two objectives: data review and quality check as well as detection of possible mutation.
  • the test described by Weir in Genetic Data Analysis II (Sinauer, 1996) has been implemented using a chi-square statistics (ldf).
  • Hardy- Weinberg equilibrium statistics were calculated separately for cases and controls data and Observed and Expected genotype frequencies were compared using a Pearson's ⁇ 2 test. A departure from Hardy- Weinberg equilibrium (HWE) in case population may indicate that a mutation had occurred, which could be responsible for increasing the risk for the disease.
  • HWE Hardy- Weinberg equilibrium
  • Additional statistics include (i) the difference between allelic frequencies in cases and in controls (the larger the difference in allelic frequency for a given SNP, the more probable is an association between the genomic region containing that SNP and the disorder), (ii) the Odds Ratio (OR) of the association and (iii) the population Attributable Risk (pAR).
  • the "chosen” allele is the allele for which the frequency is increased in cases compared to controls.
  • Mantel Haenszel test comparison of the significant findings across the 2 populations.
  • the genotypic OR allows the identification of the 'risk' genotype(s) for an associated biallelic marker. The genotypic odds ratio was calculated and Tables 2 to 5 shows the significant results of the respective genes analysed.

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Abstract

The present invention relates, generally, to methods and compositions for detecting or treating inflammatory CNS disorders, such as multiple sclerosis. The present invention more particularly discloses the identification of human genes which can be used for the diagnosis, prevention and treatment of multiple sclerosis and related disorders, as well as for the screening of therapeutically active drugs. The invention further discloses specific polymorphisms or alleles of the AUTS2, PARK2, PSEN1 and/or KCNIP4 gene that are related to multiple sclerosis, as well as diagnostic tools and kits based on these markers. The invention can be used in the diagnosis of or predisposition to, detection, prevention and/or treatment of mult iple sclerosis and related disorders.

Description

COMPOSITIONS AND METHODS FOR TREATING INFLAMMATORY CNS DISORDERS
FIELD OF THE INVENTION
The present invention relates, generally, to methods and compositions for detecting or treating inflammatory CNS disorders, such as multiple sclerosis (MS). The present invention more particularly relates to the human AUTS2, PARK2, PSENl and KCNIP4 genes, which can be used for the diagnosis, prevention and treatment of multiple sclerosis and related disorders, as well as for the screening of therapeutically active drugs. The invention further discloses specific polymorphisms or alleles of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that are related to inflammatory CNS disorders such as multiple sclerosis, as well as diagnostic tools and kits based on these markers. Thus, the invention can be used in the diagnosis or detection of the presence, risk or predisposition to, as well as in the prevention and/or treatment of multiple sclerosis and related disorders.
BACKGROUND OF THE INVENTION
Multiple sclerosis (MS) is the most known chronic inflammatory demyelinating disease of the central nervous system in humans. The onset of the disease typically occurs during ages 20 to 40. Women are affected approximately twice as often as men.
Over time, MS may result in the accumulation of various neurological disabilities. Clinical disability in MS is presumed to be a result of repeated inflammatory injury with subsequent loss of myelin and axons, leading to tissue atrophy.
MS is manifested in physical symptoms (relapses and disability progression), Central Nervous System (CNS) inflammation, brain atrophy and cognitive impairment. Presenting symptoms include focal sensory deficits, focal weakness, visual problems, imbalance and fatigue. Sexual impairment and sphincter dysfunction may occur. Approximately half of the patients with MS may experience cognitive impairment or depression.
MS is now considered to be a multi-phasic disease and periods of clinical quiescence (remissions) occur between exacerbations. Remissions vary in length and may last several years but are infrequently permanent. Four courses of the disease are individualized: relapsing-remitting (RR), secondary progressive (SP), primary progressive (PP) and progressive relapsing (PR) multiple sclerosis. More than 80% of patients with MS will initially display a RR course with clinical exacerbation of neurological symptoms, followed by a recovery that may or may not be complete {Lublin andReingold, Neurology, 1996, 46:907-911).
During RRMS, accumulation of disability results from incomplete recovery from relapses. Approximately, half of the patients with RRMS switch to a progressive course, called SPMS, 10 years after the diseased onset. During the SP phase, worsening of disability results from the accumulation of residual symptoms after exarcerbation but also from insidious progression between exacerbations {Lublin and Reingold above). 10% of MS patients have PPMS which is characterized by insidious progression of the symptoms from the disease onset. Less than 5 % of patients have PRMS and are often considered to have the same prognosis as PPMS. It is suggested that distinct pathogenic mechanisms may be involved in different patient sub-groups and have wide-ranging implications for disease classification {Lassmann et al, 2001, Trends MoI. Med., 7, 115-121; Lucchinetti et al, Curr. Opin. Neurol, 2001, 14, 259-269).
MS onset is defined by the occurrence of the first neurological symptoms of CNS dysfunction. Advances in cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI) have simplified the diagnostic process and facilitated early diagnostic {Noseworthy et al, The New England Journal of Medicine, 2000, 343, 13, 938-952). The International Panel on the Diagnosis of MS issued revised criteria facilitating the diagnosis of MS and including MRI together with clinical and para-clinical diagnostic methods {Me Donald et al, 2001, Ann. Neurol, 50:121-127).
Molecules currently used for the treatment of multiple sclerosis in its various forms may have side effects and may act only against the symptoms of the disease. Consequently, there is a strong need for new molecules without or with less associated side effects that are directed against novel targets. Therefore, there is a need to identify proteins involved in the disease, thereby providing new targets allowing new screenings for drugs, resulting in new drugs that are efficient in treatment of this serious inflammatory CNS disease and related disorders.
Furthermore, there is also a need for new diagnostic tools. Affected patients may not realize that they are ill, or they may be afraid to seek help; family members sometimes hope the problem will simply disappear or cannot persuade the patient to seek treatment; clinicians may hesitate to prescribe medications when the diagnosis is uncertain because of potential side effects. Indeed, at the first manifestation of the disease, multiple sclerosis may be difficult to diagnose with certainty. Accordingly, there is a need for new methods for detecting a susceptibility to multiple sclerosis and related disorders.
SUMMARY OF THE INVENTION
The present invention now discloses novel approaches to the diagnosis and treatment of multiple sclerosis and related disorders, as well as for the screening of therapeutically active drugs. The invention more specifically demonstrates that alterations in the AUTS2,
PARK2, PSENl and/or KCNIP4 gene are associated with the development of multiple sclerosis. AUTS2, PARK2, PSENl and/or KCNIP4, and altered forms of AUTS2, PARK2,
PSENl and/or KCNIP4 in particular, represent novel targets for therapeutic intervention against MS and related pathologies.
A first aspect of this invention thus resides in the use of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against multiple sclerosis and related disorders.
A further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis or related disorders, comprising determining the ability of a compound to bind a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, or a fragment thereof, particularly of an allele of said gene or polypeptide that is associated with multiple sclerosis or a related disorder, or a fragment thereof.
A further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis or related disorders, comprising testing for modulation of the activity of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, or a fragment thereof, particularly of an allele of said gene or polypeptide that is associated with multiple sclerosis or a related disorder, or a fragment thereof. Another aspect of this invention resides in a method of assessing the presence of or predisposition to multiple sclerosis or a related disorder in a subject, comprising determining (in vitro or ex vivo) the presence of an alteration (e.g., a susceptibility mutation or allele) in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
A further aspect of this invention relates to the use of a modulator of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, preferably an agonist thereof, for the preparation of a medicament for treating or preventing multiple sclerosis or a related disorder in a subject, as well as to corresponding methods of treatment.
The invention more specifically encompasses methods of treating multiple sclerosis or related disorders in a subject through a modulation of AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide expression or activity, preferably through an activation or restoration thereof. Such treatments use, for instance, a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide, a AUTS2, PARK2, PSENl and/or KCNIP4 DNA sequence (including antisense sequences, RNAi), antibodies against AUTS2, PARK2, PSENl and/or KCNIP4 polypeptides, ligands of AUTS2, PARK2, PSENl and/or KCNIP4 or drugs that modulate, preferably mimic or stimulate, AUTS2, PARK2, PSENl and/or KCNIP4 expression or activity. The invention particularly relates to methods of treating individuals having disease-associated alleles of the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
The invention further relates to the screening of alteration(s) associated with multiple sclerosis or related disorders in the AUTS2, PARK2, PSENl and/or KCNIP4 gene locus in patients. Such screenings are useful for diagnosing the presence, risk or predisposition to multiple sclerosis and related disorders, and/or for assessing the efficacy of a treatment of such disorders.
A further aspect of this invention includes nucleic acid probes and primers that allow specific detection of susceptibility markers in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA through selective hybridization or amplification. The invention also encompasses particular nucleic acids, vectors and recombinant cells, as well as kits or solid phase bound nucleic acids or proteins such as DNA or protein arrays or chips suitable for implementing the above detection, screening or treatment methods. In particular, the invention also discloses and encompasses markers in the AUTS2, PARK2, PSENl and/or KCNIP4 nucleic acids and polypeptides that are associated with multiple sclerosis and related disorders. Examples of such markers are more particularly selected from the markers as listed in Tables 2 to 5.
The invention can be used in the diagnosis of predisposition to, detection, prevention and/or treatment of multiple sclerosis and related disorders in any mammalian subjects, particularly human patients.
DETAILED DESCRIPTION OF THE INVENTION
The present invention stems from association studies conducted on different MS populations, using a number of random markers. The results of these studies show that the AUTS2, PARK2, PSENl and KCNIP4 genes are strongly associated with multiple sclerosis, and that new and validated (biallelic) markers located in said gene or corresponding RNAs are associated with multiple sclerosis and related disorders.
The present invention thus provides novel means and methods to identify compounds useful in the treatment of multiple sclerosis and related disorders. The invention further provides novel approaches to the detection, diagnosis and monitoring of multiple sclerosis or related disorders in a subject, as well as for genotyping of inflammatory CNS patients, in particular patients having MS.
DEFINITIONS
The term "multiple sclerosis" may be defined as in the DSM-IV classification (Diagnosis and Statistical Manual of Inflammatory CNS Disorders, Fourth Edition, American Psychiatric Association, Washington D.C., 1994).
The term "inflammatory CNS disorder" includes in particular demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases. Inflammatory CNS disorders other than MS are also called MS-related diseases herein.
As used in the present application, the term "AUT S2" designates the human AUTS2 gene, as well as variants, analogs and fragments thereof. The nucleic and amino acid sequences of a AUTS2 gene or polypeptide are available in the literature (AUTS2 GENE; AUTS2 (KIAA0442) - Gene map locus 7ql l.2 - Size: 1259 amino acids; 138981 Da. Two isoforms identified in SwissProt entry "AUTS2 HUMAN").
The human AUTS2 gene has been suggested to be implicated in autism, but prior to the present invention has not been linked to inflammatory CNS disorder such as demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases.
Sultana et al. (Genomics 80: 129-134, 2002) identified and characterized a novel gene, which they designated AUTS2, that spans the 7ql 1.2 breakpoint. AUTS2 is identical to the KIAA0442 gene identified by Ishikawa et al. (DNA Res. 4: 307-313, 1997). The predicted 1,259-amino acid protein contains 2 proline-rich domains and regions that share homology with the dwarfin family consensus sequence and with topoisomerase. It has a PY motif, several putative phosphorylation sites and N-myristoylation sites, and 2 putative N- glycosylation sites. The AUTS2 protein shares 93% amino acid identity with its murine homolog. Northern blot analysis detected strong expression of 7.5- and 8.0-kb transcripts in fetal and adult brain. AUTS2 is also strongly expressed in skeletal muscle and kidney, with lower levels in placenta, lung, and leukocytes. In fetal brain, AUTS2 is expressed in frontal, parietal, and temporal lobes, but not in the occipital lobe. Sultana et al. (2002) identified several smaller splice variants of AUTS2. By genomic sequence analysis, Sultana et al. (2002) determined that the AUTS2 gene spans 1.2 Mb and contains 19 exons.
Sultana et al. (Genomics 80: 129-134, 2002) sought to determine whether genetic variation in the AUTS2 gene contributes to idiopathic autism. DNA sequence analysis of autism subjects and controls revealed 22 biallelic polymorphic sites. For all sites, both alleles were observed in both cases and controls. Thus, no autism-specific mutation was observed. As used in the present application, the term "PARK2" designates the human PARK2 gene, as well as variants, analogs and fragments thereof. The nucleic and amino acid sequences of a PARK2 gene or polypeptide are available in the literature (PARKIN; PARK2 (PRKN - FRAGILE SITE FRA6E, INCLUDED) - Gene map locus 6q25.2-q27 - Size: 465 amino acids; 51650 Da - Localisation: cytoplasmic). 5 iso forms have been identified in SwissProt entry PRKN2 HUMAN.
The human PARK2 gene has been linked to Parkinson disease, but not inflammatory CNS disorder such as demyelinating inflammatory CNS disorders, such as for example, MS, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM) or other related diseases.
Autosomal recessive juvenile parkinsonism maps to 6q25.2-q27, as indicated by linkage to markers D6S305 and D6S253. The former was deleted in 1 Japanese patient with PDJ (Matsumine et al., 1997). By positional cloning within this microdeletion, a cDNA clone of 2,960 bp with a 1,395-bp open reading frame was isolated, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and with a ring finger motif at the carboxy terminus. A 4.5-kb transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from 1 of the exon-4-deleted patients. Mutations in the newly identified gene appeared to be responsible for the pathogenesis of PDJ and, therefore, the protein product was designated 'parkin.'
PARK2 forms an E3 ubiquitin ligase complex with UBE2L3 or UBE2L6 (part of a SCF- like complex, consisting of PARK2, CULl and FBXW7). PARK2 interacts with SNCAIP and binds to the C2A and C2B domains of SYTl 1. It interacts and regulates the turnover of SEPT5. It is also part of a complex, including STUBl, HSP70 and GPR37. The amount of STUBl in the complex increases during ER stress. STUBl promotes the dissociation of HSP70 from PARK2 and GPR37, thus facilitating PARK2-mediated GPR37 ubiquitination. HSP70 transiently associates with unfolded GPR37 and inhibits the E3 activity of PARK2, whereas, STUBl enhances the E3 activity of PARK2 through promotion of dissociation of HSP70 from PARK2-GPR37 complexes. PARK2 interacts with PSMD4. PARK2 is cytoplasmic and co-localizes with STYIl in neutrites. PARK2 co-localizes as well with SNCAIP in brainstem Lewy bodies. PARK2 auto-ubiquitinates in an E2-dependent manner leading to its own degradation. PARK2 is S-nitrosylated. The inhibition of PARK2 ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in PD by impairing the ubiquitination of PARK2 substrates. The parkin locus (PRKN), adjacent to the 6q telomere is hyper-recombinable and lies within FRA6E, the third most common fragile site in tumor tissue.
Defects in park2 are a cause of parkinson's disease (pd). PD is a complex, multifactorial disorder that typically manifests after the age of 50 years, although early- onset cases (before 50 years) are known. PD is characterized by bradykinesia, resting tremor, muscular rigidity and postural instability, as well as by a clinically significant response to treatment with levodopa. The pathology of PD involves the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (intraneuronal accumulations of aggregated proteins), in surviving neurons in various areas of the brain. Defects in park2 are also a cause of autosomal recessive juvenile parkinsonism (ar-jp); also known as early- onset parkinsonism with diurnal fluctuation (EPDF), autosomal recessive juvenile Parkinson's disease (PDJ) or Parkinson's disease type 2 (PARK2). AR-JP is symptomatically different in several aspects from idiopathic Parkinson's disease, although classic symptoms such as bradykinesia, rigidity and tremor are present. Additional clinical features include early DOPA-induced dyskinesia, diurnal fluctuation of the symptoms, sleep benefit, dystonia and hyper-reflexia. AR-JP is usually characterized by onset before 40, with a mean age at onset of 23.2 years. Pathologically, AR-JP patients show loss of dopaminergic neurons in the substantia nigra, similar to that seen in Parkinson's disease; however, Lewy bodies (intraneuronal accumulations of aggregated proteins) are absent. Defects in PARK2 may be involved in the development and/or progression of ovarian cancer.
As used in the present application, the term "PSENl" designates the human PSENl gene, as well as variants, analogs and fragments thereof. The nucleic and amino acid sequences of a PSENl gene or polypeptide are available in the literature. The PSENl gene (PRESENILIN 1; PSENl (PSl, S182) - Gene map locus 14q24.3 - Size: 467 amino acids; 52667 Da) has previously been related to Alzheimers disease, but not to inflammatory CNS disorders, such as MS. 6 isoforms have been identified (see SwissProt etnry PSNl HUMAN). Heterogeneous proteolytic processing generates N-terminal and C- terminal fragments of approximately 35 and 20 kDa, respectively. PSENl is phosphorylated on serine residues.
By linkage mapping, Sherrington et al. (1995) defined a minimal cosegregating region containing the candidate gene for early-onset Alzheimer disease type 3, which had been linked to chromosome 14q24.3. Of 19 different transcripts isolated, 1 transcript, designated S 182 by them, corresponded to a novel gene that encoded a 467-amino acid protein. Human and murine amino acid sequences shared 92% identity. Northern blot analysis identified a major 3-kb transcript expressed in most regions of the human brain and in several peripheral tissues. Structural analysis predicted an integral membrane protein with at least 7 transmembrane helical domains. The Alzheimer's Disease Collaborative Group (1995) isolated full-length cDNA clones for what they referred to as the PSl gene. The Alzheimer's Disease Collaborative Group (1995) determined that the open reading frame of PSl is encoded by 10 exons. They concluded that the PS2 gene (600759), located on chromosome 1, has a very similar gene structure. PSENl is a probable catalytic subunit of the gamma-secretase complex, an endoprotease complex that catalyzes the intramembrane cleavage of integral membrane proteins such as Notch receptors and APP (beta-amyloid precursor protein). It requires the other members of the gamma-secretase complex to have a protease activity. It may play a role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane. PSENl is also believed to regulate epithelial- cadherin function.
As used in the present application, the term "KCNIP4" designates the human KCNIP4 gene, as well as variants, analogs and fragments thereof. The nucleic and amino acid sequences of a KCNIP4 gene or polypeptide are available in the literature (POTASSIUM CHANNEL-INTERACTING PROTEIN 4 (KCNIP4; KCHIP4, CALSENILIN-LDCE PROTEIN; CALP) - Gene map locus 4ρl5.3). 4 alternative transcripts of KCNIP4 have been identified (REFSEQ proteins NP_079497.2, NP_671710.1, NP_671711.1, NP_671712.1). Heterogeneous proteolytic processing generates N-terminal and C-terminal fragments of approximately 35 and 20 kDa, respectively. KCNIP4 is phosphorylated on serine residues.
It is known that K channel-interacting proteins, such as KCNIP4, specifically modulate the activity of Kv4 A-type potassium channels (KCNDl). Kv4 A-type channels are known to contribute to the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart (Holmqvist et al., Proc. Nat. Acad. Sci. 99: 1035-1040, 2002). However, prior to this invention KCNIP4 has not been linked to inflammatory CNS disorders such as for example MS.
Holmqvist et al. (2002) cloned mouse Kcnip4, which they designated Kchip4. By analyzing human and rodent databases, they identified several KCNIP4 splice variants that differ from each other mostly in the N-terminal domain. Human and mouse variants that include an N-terminal 34-amino acid K channel inactivation suppressor (KIS) domain share 100% amino acid identity. Northern blot analysis of rat tissues detected Kcnip4 expression in brain, but not in heart or any other tissue examined. RT-PCR of mouse tissues detected expression of 2 splice variants in brain, but not in atrium or ventricle.
Holmqvist et al. (2002) determined that coexpression of mouse Kcnip4 with Kv4 alpha subunits abolished fast inactivation of the Kv4 current in various cell types, including rat cerebellar granule neurons. The KIS domain of Kcnip4 delayed Kv4.3 (KCND3) opening, but, once the channel opened, it disrupted rapid inactivation and slowed Kv4.3 closing.
By yeast 2-hybrid analysis, Morohashi et al. (Biol. Chem. 277: 14965-14975, 2002) determined that KCNIP4 interacts with the C termini of PS2 and PSl. By Ca(2+) overlay assay, they determined that the EF hand motif of KCNIP4 binds calcium. Using mutation analysis and coimmunoprecipitation experiments, they showed that the EF hand motif mediates the interaction between KCNIP4 and rat Kv4. Overexpression of KCNIP4 did not alter the metabolism or stability of PS, but overexpression of KCNIP4 with rat Kv4.2 (KCND2;) reconstituted the features of A-type K+ currents. Morohashi et al. (2002) concluded that KCNIP4 is a novel EF hand protein that interacts with both PS and Kv4.
The term "gene" shall be construed to include any type of coding nucleic acid region, including genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semisynthetic DNA, any form of corresponding RNA (e.g., mRNA), etc., as well as non coding sequences, such as introns, 5'- or 3 '-untranslated sequences or regulatory sequences (e.g., promoter or enhancer), etc. The term gene particularly includes recombinant nucleic acids, i.e., any non naturally occurring nucleic acid molecule created artificially, e.g., by assembling, cutting, ligating or amplifying sequences. A gene is typically double-stranded, although other forms may be contemplated, such as single-stranded. Genes may be obtained from various sources and according to various techniques known in the art, such as by screening DNA libraries or by amplification from various natural sources. Recombinant nucleic acids may be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. The term "gene" may comprise any and all splicing variants of said gene.
A fragment of a gene designates any portion of at least about 8 consecutive nucleotides of a sequence of said gene, preferably at least about 15, more preferably at least about 25 nucleotides, further preferably of at least 35, 50, 75, 100, 150, 200 or 300 nucleotides. Fragments include more particularly all possible nucleotide length between 8 and 500 nucleotides, preferably between 15 and 300, more preferably between 25 and 200.
A AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide designates any protein or polypeptide encoded by a AUTS2, PARK2, PSENl and/or KCNIP4 gene as disclosed above, respectively. In this respect, the term "polypeptide" designates, within the context of this invention, a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. In particular a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide also denotes a polypeptide, which is specific fragment of AUTS2, PARK2, PSENl and/or KCNIP4 of at least 8, 15, 20, 50, 100, 250, 300 or 350 amino acids in length. This term also does not specify or exclude post-translational or post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
Fusion proteins are useful for generating antibodies against a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide and for use in various assay systems. For example, fusion proteins can be used to identify proteins, which interact with portions of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide. Protein affinity chromatography or library- based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
A AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 25, 50, 75, 100, 150, 200, 300, 350 or 372 contiguous amino acids of SEQ ID NO: 2. The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include beta- galactosidase, beta-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide-encoding sequence and the heterologous protein sequence, so that the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide can be cleaved and purified away from the heterologous moiety.
A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences for AUTS2, PARK2, PSENl and/or KCNIP4 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
The term "treat" or "treating" as used herein is meant to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition. The term "treatment" as used herein also encompasses the term "prevention of the disorder", which is, e.g., manifested by delaying the onset of the symptoms of the disorder to a medically significant extent. Treatment of the disorder is, e.g., manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.
The terms "modulated" or "modulation" or "regulated" or "regulation" as used herein refer to both upregulation [i.e., activation or stimulation (e.g., by agonizing or potentiating)] and downregulation [i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)].
As used interchangeably herein, the term "oligonucleotides", and "polynucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term "nucleotide" as used herein as an adjective to describe compounds comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term "nucleotide" is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a compound, or individual unit in a larger nucleic acid compound, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term "nucleotide" is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO95/04064, the disclosure of which is incorporated herein by reference. However, the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art. The term "isolated" requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
The term "primer" denotes a specific oligonucleotide sequence, which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase. Typical primers of this invention are single- stranded nucleic acid molecules of about 6 to 50 nucleotides in length, more preferably of about 8 to about 40 nucleotides in length, typically of about 16 to 25. The Tm is typically of about 60°C or more. The sequence of the primer can be derived directly from the sequence of the target gene. Perfect complementarity between the primer sequence and the target gene is preferred, to ensure high specificity. However, certain mismatch may be tolerated.
The term "probe" denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified. Probes of this invention typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 750, more preferably of between 15 and 600, typically of between 20 and 400. The sequence of the probes can be derived from the sequences of the AUTS2, PARK2, PSENl and/or KCNIP4 gene sequence. The probe may contain nucleotide substitutions and/or chemical modifications, e.g., to increase the stability of hybrids or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, etc.
The terms "complementary" or "complement thereof are used herein to refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
As used herein, the term "non-human animal" refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term "animal" is used to refer to any vertebrate, preferable a mammal. Both the terms "animal" and "mammal" expressly embrace human subjects unless preceded with the term "non-human".
The terms "trait" and "phenotype" are used interchangeably herein and refer to any clinically distinguishable, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Typically the terms "trait" or "phenotype" are used herein to refer to symptoms of, or susceptibility to inflammatory CNS disorder; or to refer to an individual's response to an agent acting on inflammatory CNS disorder; or to refer to symptoms of, or susceptibility to side effects to an agent acting on inflammatory CNS disorder.
As used herein, the term "allele" refers to one of the variant forms of a biallelic or multiallelic marker, differing from other forms in its nucleotide sequence. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
The term "polymorphism" as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. "Polymorphic" refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A "polymorphic site" is the locus at which the variation occurs. A polymorphism may comprise a substitution, deletion or insertion of one or more nucleotides. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. A "single nucleotide polymorphism" (SNP) refers to a sequence polymorphism differing in a single base pair.
DETECTION, DIAGNOSIS AND THERAPY
The present invention provides novel means and methodologies for detecting or diagnosing multiple sclerosis and related disorders in a human subject. The present methods may be implemented at various development stages of said pathologies, including early, pre- symptomatic stages, and late stages, in adults, children and pre-birth. Furthermore, the invention is suited to determine the prognosis, to assess a predisposition to or a risk of development of pathology, to characterize the status of a disease or to define the most appropriate treatment regimen for a patient.
A particular object of this invention resides in a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence of an alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
Another object of this invention relates to methods of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder, the methods comprising detecting the presence of an alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
Current medications for MS which are disease modifying treatments, i.e. modifying the course of MS, modulate or suppress the immune system. Medications for MS within the meaning of the present invention include the four FDA approved immunomodulating agents for RRMS: three beta interferons (Betaseron®, Berlex; Avonex®, Biogen; Rebif®, Serono) and Glatimarer Acetate (Copaxone®, Amgen). Medications for MS within the meaning of the present invention also include the FDA approved immunosuppressing drug for worsening MS, Mitoxantrone (Novantrone®, Amgen). The terms "interferon (IFN)" and "interferon-beta (IFN-beta)", as used herein, are intended to include fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments.
IFN-beta suitable in accordance with the present invention is commercially available e.g. as Rebif® (Serono), Avonex® (Biogen) or Betaferon® (Schering). The use of interferons of human origin is also preferred in accordance with the present invention. The term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof.
Rebif® (recombinant human interferon-) is the latest development in interferon therapy for multiple sclerosis (MS) and represents a significant advance in treatment. Rebif® is interferon (IFN)-beta Ia, produced from mammalian cell lines. It was established that interferon beta- Ia given subcutaneously three times per week is efficacious in the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS). Interferon beta- Ia can have a positive effect on the long-term course of MS by reducing number and severity of relapses and reducing the burden of the disease and disease activity as measured by MRI.
The dosing of IFN-β in the treatment of relapsing-remitting MS according to the invention depends on the type of IFN-β used.
In accordance with the present invention, where IFN is recombinant IFN-β Ib produced in E. CoIi, commercially available under the trademark Betaseron, it may preferably be administered sub-cutaneously every second day at a dosage of about of 250 to 300 g or 8 MIU to 9.6 MIU per person.
In accordance with the present invention, where IFN is recombinant IFN-β Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Avonex, it may preferably be administered intra-muscularly once a week at a dosage of about of 30g to 33 g or 6 MIU to 6.6 MIU per person.
In accordance with the present invention, when IFN is recombinant IFN-β Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Rebif, it may preferably be administered sub-cutaneously three times a week (TIW) at a dosage of 22 to 44 g or 6 MIU to 12 MIU per person. As will be discussed below in more details, the alteration ("susceptibility alteration") in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide may be any susceptibility marker in said gene or polypeptide, i.e., any nucleotide or amino acid alteration associated to multiple sclerosis or a related disease.
An alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertion(s) in the coding and/or non- coding region of the gene, either isolated or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene. Typical deletions affect small regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene. Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene. Rearrangements include for instance sequence inversions. An alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene may also be an aberrant modification of the polynucleotide sequence, such as of the methylation pattern of the genomic DNA, allelic loss of the gene or allelic gain of the gene. The alteration may be silent (i.e., create no modification in the amino acid sequence of the protein), or may result, for instance, in amino acid substitutions, frameshift mutations, stop codons, RNA splicing, e.g. the presence of a non-wild type splicing pattern of a messenger RNA transcript, or RNA or protein instability or a non-wild type level of the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide. Also, the alteration may result in the production of a polypeptide with altered function or stability, or cause a reduction or increase in protein expression levels. Typical alterations are single nucleotide substitutions.
The chromosomal SNP positions ("pos") given in the present patent application are based on NCBI human genome Build 35. These position show associated alleles which are preferred markers of the invention. The sites of the SNPs ("site") are further identified by unique Affymetrix SNP identifiers referring to the Affymetrix genechip human mapping IOOK set. These Affymetrix identifiers refer to unique oligonucleotides of 25bp length which allow for an identification of the respective SNP independent of the sequence of a specific genome build/version.
In this regard, the present invention now discloses several markers or mutations in the AUTS2 gene, which are associated with multiple sclerosis. These mutations are reported in tables 2a and 2b. Preferred genetic alterations are disclosed in table 2a below. Most preferred alterations are disclosed in table 2b below.
In summary, the association results of the single biallelic marker frequency analysis show that the AUTS2 gene is associated with multiple sclerosis.
Table 2b
R= Rennes population, H= Huddinge population, S= Seracare population.
A preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 2a in the AUT S2 gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 2b, or any combination thereof.
A preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 2a in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
It is understood that one or more markers, as well as any combination thereof are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject. Furthermore, the additional presence of an associated allele in the PARK2, PSENl and/or KCNIP4 gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject. In this regard, the present invention now discloses several markers or mutations in the PARK2 gene, which are associated with multiple sclerosis. These mutations are reported in table 3 (table 3a and 3b). Preferred genetic alterations are disclosed in table 3a below. Most preferred alterations are disclosed in table 3b below.
In summary, the association results of the single biallelic marker frequency analysis show that the PARK2 gene is associated with multiple sclerosis.
Table 3b
R= Rennes population, H= Huddinge population, S= Seracare population.
A preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 3a in the PARK2 gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 3b, or any combination thereof.
A preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 3a or 3b in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
It is understood that one or more markers, as well as any combination thereof are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject. Furthermore, the additional presence of an associated allele in the AUTS2, PSENl and/or KCNIP4 gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
In this regard, the present invention now discloses several markers or mutations in the PSENl gene, which are associated with multiple sclerosis. These mutations are reported in table 4 (table 4a and table 4b). Preferred genetic alterations are disclosed in table 4a below. Most preferred alterations are disclosed in table 4b below.
In summary, the association results of the single biallelic marker frequency analysis show that the PSENl gene is associated with multiple sclerosis.
Table 4a
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Table 4b
R= Rennes population, H= Huddinge population, S= Seracare population.
A preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 4a in the PSENl gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 4b, or any combination thereof.
A preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 4a or 4b in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
It is understood that one or more markers, as well as any combination thereof are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject. Furthermore, the additional presence of an associated allele in the AUTS2, PARK2, and/or KCNIP4 gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
In this regard, the present invention now discloses several markers or mutations in the KCNIP4 gene, which are associated with multiple sclerosis. These mutations are reported in table 5 (tables 5a and 5b). Preferred genetic alterations are disclosed in table 5a below. Most preferred alterations are disclosed in table 5b below.
In summary, the association results of the single biallelic marker frequency analysis show that the KCNIP4 gene is associated with multiple sclerosis.
table 5a
RENNES HUDDINGE SERACARE site chr pos Chosen Allele Allelic P- OR Genot HWE cases Chosen Allele AII_Pvalue OR Genot P' HWE cases Chosen Allele AII_Pvalue OR Genot_P HWE
Allele frequency value P- P-value Allele frequency value P-value Allele frequency value cases difference value difference difference Pvalue
SNP_A-1735559 4 20380352 G 0,047 0,0484 1,3 0,0293 0,014
SNP A-1735670 4 20380609 A 0,014 0,36 1,2 0,18 0,49 A 0,037 0,055 1,4 0,12 0,47 T 0,007 0,71 1,1 0,31 1
SNP.A-1741904 4 20403362 G 0,03 0,00506 2,7 0,00219 1
SNP.A-1742036 4 20403733 G 0,019 0,21 1,3 0,096 0,49 G 0,033 0,089 1,4 0,22 0,54 G 0,006 0,74 1,1 0,34 0,7
SNP_A-1730005 4 20539255 G 0,053 0,022 1,4 0,053 0,53
SNP.A-1736131 4 20542738 C 0,047 0,0435 1,4 0,12 0,75
SNP.A-1693268 4 20566766 A 0,032 0,0091 2 0,012 1 T 0,035 0,0301 1,7 0,03475 0,052
SNP A-1666023 4 20604766 C 0,062 0,0316 1,3 0,087 0,31
SNP.A-1727836 4 20772414 G 0,055 0,00796 1,5 0,03 0,8 G 0,088 0,000106 1,8 3.4E-05 0,0111
SNP.A-1727928 4 20772468 C 0,053 0,0114 1,45 0,04 0,91 C 0,088 0,00014 1,82 1.41E-05 4.20E-03 C 0,01 0,7 1,08 0,77 0,71 κ>
SNP.A-1729529 4 20773096 A 0,054 0,0115 1,5 0,04 0,53 A 0,089 0,000132 1,8 1.3E-05 0,000347
SNP.A-1688310 4 20789318 A 0,052 0,0322 1,4 0,1 0,51 G 0,052 0,04 1,3 0,11 0,49
SNP.A-1723543 4 20851888 A 0,015 0,36 1,2 0,034 0,5 A 0,043 0,0297 1,5 0,0183 0,52
SNP.A-1723647 4 20851922 A 0,071 0,00892 1,3 0,001 0,043
SNP.A-1677329 4 20924264 A 0,017 0,31 1,2 0,03114 0,38
SNP_A-1667495 4 21155517 G 0,018 0,51 1,1 0,29 0,0000281
SNP_A-1661543 4 21221101 G 0,068 0,0344 1,3 0,0211 0,48
SNP_A-1655856 4 21295077 C 0,019 0,42 1,1 0,57 0,0098
SNP_A-1725873 4 21369994 C 0,056 0,0487 1,3 0,074 0,16
SNP_A-1686638 4 21441030 C 0 1 1 0,31 0,38
SNP_A-1668671 4 21559839 T 0,052 0,0196 1,4 0,074 0,13
SNP_A-1714237 4 21594415 T 0,049 0,041 1,3 0,13 0,55
SNP_A-1723255 4 21641733 C 0,038 0,0274 1,6 0,001852 0,39
SNP_A-1676034 4 21649447 C 0,076 0,00554 1,4 0,00449 0,81
Table 5b
MH pvalue MH pvalue name chromosome position R+H R+H+S
SNP A-1667495 4 21155517 0,0856 0,0212
SNP_A-1714237 4 21594415 0,00479 0,011
SNP A-1723543 4 20851888 0,0283 0,0549
SNP_A-1723647 4 20851922 0,0369 0,0219
SNP_A-1727836 4 20772414 0,00000439 0,0000345
SNP A-1727928 4 20772468 0,00000665 0,0000643
SNP_A-1729529 4 20773096 0,00000772 0,00000958
SNP A-1735670 4 20380609 0,0409 0,14
SNP A-1742036 4 20403733 0,0361 0,049
R= Rennes population, H= Huddinge population, S= Seracare population.
A preferred embodiment of the present invention comprises the detection of the presence of a marker as disclosed in Table 5a in the KCNIP4 gene or RNA sequence of a subject, more particularly the detection of at least one marker as disclosed in Table 5b, or any combination thereof.
A preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated allele according to table 5a or 5b in a sample from the subject, the presence of the associated allele being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
It is understood that one or more markers, as well as any combination thereof are indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject. Furthermore, the additional presence of an associated allele in the AUTS2, PARK2, and/or PSENl gene as disclosed herein is indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject. Now that the association between AUTS2, PARK2, PSENl and/or KCNIP4 and multiple sclerosis or related diseases has been established by the inventors, it should be understood that additional susceptibility markers can be identified within said gene or polypeptide, e.g., following the methodology disclosed in the examples.
The presence of an alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene may be detected by any technique known per se to the skilled artisan (reviewed by Kwok et al., 2003), including sequencing, pyrosequencing, selective hybridisation, selective amplification and/or mass spectrometry including matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) (Gut et al., 2004). In a particular embodiment, the alteration is detected by selective nucleic acid amplification using one or several specific primers. The alteration is detected by selective hybridization using one or several specific probes.
Further techniques include gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing; fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping; rolling circle amplification and Invader assays as well as DNA chip-based microarray and mass spectrometry genotyping technologies (Shi et al., 2001).
Furthermore, RNA expression of altered genes can be quantified by methods known in the art such as subtractive hybridisation, quantitative PCR, TaqMan, differential display reverse transcription PCR, serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips. Particular methods include allele-specific oligonucleotide (ASO), allele-specific amplification, fluorescent in situ hybridization (FISH) Southern and Northern blot, and clamped denaturing gel electrophoresis. Protein expression analysis methods are known in the art and include 2-dimensional gel- electrophoresis, mass spectrometry and antibody microarrays (Freeman et al., 2004 and Zhu et al., 2003).
Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing may be performed on the complete gene or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.
Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA). These techniques can be performed using commercially available reagents and protocols. A preferred technique is allele-specific PCR.
Nucleic acid primers useful for amplifying sequences from the AUTS2, PARK2, PSENl and/or KCNIP4 gene are able to specifically hybridize with a portion of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that either flanks or overlaps with an alteration, such as a susceptibility marker. The primer sequence overlaps with the alteration when said alteration is contained within the sequence of the AUTS2, PARK2, PSENl and/or KCNIP4 gene to which the primer hybridizes. The primer sequence flanks the alteration when the primer hybridizes with a portion of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that is preferably located at a distance below 300 bp of said alteration, even more preferably below 250, 200, 150, 100, 50, 40, 30 or 20 bp from said alteration. Preferably, the primer hybridizes with a portion of the AUTS2, PARK2, PSENl and/or KCNIP4 gene that is at 5, 4, 3, 2, 1 bp distance or immediately adjacent to said alteration.
The invention also relates to 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 multiple sclerosis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder. According to another embodiment of the present invention, the methods involve the use of a nucleic acid probe specific for a AUTS2, PARK2, PSENl and/or KCNIP4 or altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA, followed by the detection of the presence of a hybrid. The probe may be used in suspension or immobilized on a substrate or support. The probe is typically labelled to facilitate detection of hybrids.
In this respect, a specific object of this invention is a nucleic acid probe complementary to and specific for a region of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA that carries an alteration as described in Table 2a to 5a and preferably Table 2b to 5b. The probes of the present invention are, more preferably, capable of discriminating between an altered and non-altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA sequence, i.e., they specifically hybridise to a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA carrying a particular alteration as described above, and essentially do not hybridise under the same hybridization conditions or with the same stability to a AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA lacking said alteration.
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 multiple sclerosis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder.
The detection methods can be performed in vitro, ex vivo or in vivo, preferably in vitro or ex vivo. They are typically performed on a sample from the subject, such as any biological sample containing nucleic acids or polypeptides. Examples of such samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma, saliva, urine, seminal fluid, etc. The sample may be collected according to conventional techniques and used directly for diagnosis or stored. In particular, they may be obtained by non-invasive methods, such as from tissue collections. The sample may be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments include, for instant, lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, etc. Also, the nucleic acids and/or polypeptides may be pre- purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. Considering the high sensitivity of the claimed methods, very few amounts of sample are sufficient to perform the assay.
The sample is typically contacted with probes or primers as disclosed above. Such contacting may be performed in any suitable device, such as a plate, tube, well, glass, etc.
The contacting may performed on a substrate coated with said specific reagents, such as a nucleic acid array. The substrate may be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids of the sample.
The finding of an altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or RNA or polypeptide in the sample is indicative of the presence, predisposition or stage of progression of multiple sclerosis or a related disorder in the subject. Typically, one only of the above-disclosed markers is assessed, or several of them, in combination(s).
In another aspect of the invention there is provided a method of treating multiple sclerosis or a related disorder in a subject in need of such treatment comprising of administering to the subject a compound that modulates the synthesis, expression or activity of one or more of the genes or gene products of the genes listed in Table 2a to 5a and preferably Table 2b to 5b in a therapeutically effective amount so that at least one symptom of the multiple sclerosis or a related disorder is ameliorated.
In another aspect of the invention there is provided a method of treating multiple sclerosis or a related disorder in a subject in need of such treatment wherein the subject has a susceptibility alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene comprising of administering to the subject a therapeutically effective amount of a medication for MS, such as for example interferon-beta, preferably interferon-beta Ia.
Preferably the susceptibility alteration is selected from one or more of the susceptibility markers listed in Table 2a to 5a and preferably Table 2b to 5b. Preferably the susceptibility alteration is located within the 3' or 5' region of the AUTS2, PARK2, PSENl and/or KCNIP4 gene. Preferably the susceptibility susceptibility alteration is a single nucleotide mutation.
In another aspect of the invention there is provided a kit for the identification of a genetic polymorphism pattern at the AUTS2, PARK2, PSENl and/or KCNIP4 gene associated with increased risk of the presence of or predisposition to multiple sclerosis or a related disorder in a subject, said kit comprising:
(a) DNA sample collecting means, and
(b) means for determining a genetic polymorphism pattern for the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
DRUG SCREENING
As indicated above, the present invention also provides novel targets and methods for the screening of drug candidates or leads. These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
In this regard, a particular object of this invention resides in the use of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide as a target for screening candidate drugs for treating or preventing multiple sclerosis or a related disorder.
Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
A further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide with a candidate compound, and selecting compounds that bind said AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide at the surface of said cells and/or that modulate the activity of the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide. A "biologically active" compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a neuroactive compound, and further preferably a compound that can be used for treating multiple sclerosis or a related disorder, or as a lead to develop drugs for treating multiple sclerosis or a related disorder. A "biologically active" compound preferably is a compound that modulates the activity of AUTS2, PARK2, PSENl and/or KCNIP4.
The above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of multiple sclerosis or a related disorder, such as an animal model.
Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to multiple sclerosis or a related disorder in a subject. The determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc. For in vitro binding assays, the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
Modulation of activity includes, without limitation, stimulation of the surface expression of the AUTS2, PARK2, PSENl and/or KCNIP4 receptor, modulation of multimerization of said receptor (e.g., the formation of multimeric complexes with other sub-units), etc. The cells used in the assays may be any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide) or any cell that expresses an endogenous AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide. Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
Preferred selected compounds are agonists of AUTS2, PARK2, PSENl and/or KCNIP4, i.e., compounds that can bind to AUTS2, PARK2, PSENl and/or KCNIP4 and mimic the activity of an endogenous ligand thereof.
In a particular embodiment, the screening assays of the present invention use, either alone or in addition to another AUTS2, PARK2, PSENl and/or KCNIP4 sequence, an altered AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, particularly a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide having a mutation as listed in Table 2a to 5a and preferably Table 2b to 5b, respectively.
A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a AUTS2, PARK2, PSENl and/or KCNIP4 gene according to the present invention and determining the ability of said test compound to modulate the expression of said AUTS2, PARK2, PSENl and/or KCNIP4 gene, preferably to stimulate expression thereof.
In another embodiment, this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a AUTS2, PARK2, PSENl and/or KCNIP4 gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene. In another embodiment, this invention relates to the use of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide or fragment thereof, whereby the fragment is preferably a AUTS2, PARK2, PSENl and/or KCNIP4 gene-specific fragment, for isolating or generating an agonist or stimulator of the AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide for the treatment of multiple sclerosis or a related disorder, wherein said agonist or stimulator is selected from the group consisting of:
1. a specific antibody or fragment thereof including a) a chimeric, b) a humanized or c) a fully human antibody as well as
2. a bispecific or multispecific antibody,
3. a single chain (e.g. scFv) or
4. single domain antibody, or
5. a peptide- or non-peptide mimetic derived from said antibodies or 6. an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
The generation of peptide- or non-peptide mimetics from antibodies is known in the art (Saragovi et al., 1991 and Saragovi et al., 1992). Anticalins are also known in the art (Vogt et al., 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224.
Furthermore, the test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations. PHARMACEUTICAL COMPOSITIONS AND THERAPY
The present invention now discloses novel approaches to the treatment of multiple sclerosis and related disorders by modulating the activity or expression of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide. More particularly, the present invention provides the first evidence of a correlation between said gene and said diseases in human subjects, and allows the design of novel therapeutic approaches based on a modulation, preferably a stimulation or increase of a AUTS2, PARK2, PSENl and/or KCNIP4 activity.
In this regard, a particular object of this invention resides in the use of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide, or a nucleic acid encoding the same, for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
A further object of this invention resides in the use of a modulator of AUTS2, PARK2, PSENl and/or KCNIP4 for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject. Most preferably, the modulator is an agonist or activator of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
In a preferred embodiment, the agonist is a natural ligand of AUTS2, PARK2, PSENl and/or KCNIP4, or an antibody, such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non-peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), that selectively binds AUTS2, PARK2, PSENl and/or KCNIP4.
In another embodiment, the modulator is an inhibitor or antagonist of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide. A further object of this invention resides in a pharmaceutical composition comprising a nucleic acid encoding a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide or a vector encoding the same, and a pharmaceutically acceptable carrier or vehicle.
The above uses or compositions are particularly suited for treating or preventing multiple sclerosis or a related disorder in a subject presenting an alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide, particularly in a subject presenting a marker as described in Table 2a to 5a and preferably Table 2b to 5b, respectively.
Another object of this invention is an isolated or recombinant AUTS2, PARK2, PSENl and/or KCNIP4 gene or a fragment thereof, wherein said gene or fragment comprises a marker selected Table 2a to 5a and preferably Table 2b to 5b, respectively.
The invention also relates to any vector comprising a nucleic acid as defined above. The vector may be any plasmid, phage, virus, episome, artificial chromosome, and the like. In a particular embodiment, the vector is a recombinant virus. Viral vectors may be produced from different types of viruses, including without limitation baculoviruses, retroviruses, adenoviruses, AAVs, etc., according to recombinant DNA techniques known in the art.
The recombinant virus is typically replication-defective, even more preferably selected from El- and/or E4-defective adenoviruses, Gag-, pol- and/or env-defective retroviruses and Rep- and/or Cap-defective AAVs. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in
WO95/14785, WO96/22378, US5,882,877, US6,013,516, US4.861.719, US5,278,056 and
WO94/19478.
A further aspect of this invention is a recombinant host cell comprising a vector or a nucleic acid as defined above. The recombinant cell may be any prokaryotic or eukaryotic cells as discussed above. The recombinant cell preferably expresses a recombinant AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide at its surface. A preferred embodiment of the invention is the use of an activator or agonist of AUTS2, PARK2, PSENl and/or KCNIP4 or a receptor comprising AUTS2, PARK2, PSENl and/or KCNIP4 in the preparation of a medicament for the treatment of multiple sclerosis or a related disorder.
A particularly preferred embodiment of the invention is the use of an activator or agonist of AUTS2, PARK2, PSENl and/or KCNIP4 or a receptor comprising AUTS2, PARK2, PSENl and/or KCNIP4 in the preparation of a medicament for the treatment of multiple sclerosis or a related disorder wherein the activator or agonist is an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin).
The invention also relates to a method of treating or preventing multiple sclerosis or a related disorder in a subject, the method comprising administering to said subject a compound that modulates, preferably that activates or mimics, expression or activity of a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide as defined above.
A particular embodiment of the present invention resides in a method of treating or preventing multiple sclerosis or a related disorder in a subject, the method comprising (i) detecting in a sample from the subject the presence of an alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide as defined above and (ii) administering to said subject an agonist of AUTS2, PARK2, PSENl and/or KCNIP4. Preferably, said alteration is selected from the group consisting of an alteration as disclosed in Table 2a to 5a and preferably Table 2b to 5b, respectively.
Further aspects and advantages of the present invention will be disclosed in the following section, which should be regarded as illustrative and not limiting the scope of the present application. EXAMPLES
Example 1: Materials and Methods
1.1 Collections of patients and DNA banking - Subjects
The study comprised three collections of unrelated patients with multiple sclerosis (MS) and unrelated healthy controls recruited from the neurological Department of Rennes (France: 314 cases; 353 controls), Huddinge (Sweden: 279 cases; 301 controls) hospitals and SeraCare (USA: 289 cases; 289 controls). Table 1 provides a summary for the description and stratification study of the different collections.
Informed consent was given by each individual participating in the study, according to the Helsinki Convention (1964).
The following variables were recorded for each patient: sex, ethnic background, family history with regards to MS, diagnostic category, disease course, age at disease onset, results of cerebrospinal fluid (CSF) and Magnetic Resonance Imaging (MRI) examination, Expanded Disability Status scale (EDSS) score and disease duration at last inter-relapse clinical examination.
Disease courses were classified as relapsing-remitting (RR), relapsing-secondary progressive (SP), or primary-progressive (PP) as follows:
• RR: relapses with full recovery or with a residual deficit and lack of progression between relapses; • SP: initial RR MS followed by progression;
• PP: Disease progression from onset
Selected for this study were patients with Primary progressive type, remitting-relapsing type or secondary progressive type MS, who have been diagnosed as MS according to the criteria of Mc Donald et al. (2001).
Each subject was assessed clinically by the Kurtzke Expanded Disability Status Scale (EDSS) (Kurtzke, 1983), using the latest data available.
Rennes collection: Each patient and control subject included in the analysis had to be born in Bretagne, France as well as their parents and grand-parents.
The female / male ratio in the patient group was 2.14 (214 Females & 100 Males) with a mean age of 44 [19;68] years and in the control group 2.07 (241 Females & 116 Males) with a mean age of 35 [18;56] years.
Huddinge collection:
All participants in this study were drawn from a homogeneous population of Huddinge, Sweden.
The female / male ratio in the patient group was 2.4 (196 Females & 83 Males) with a mean age of 47 [22;75].
The control group in Huddinge collection included 301 (214 Females & 87 Males) healthy volunteers and the Female / male ratio was 2.5. Ages ranged from 22 to 73 years with a mean age of 47 years.
Seracare collection:
All the subjects included in the study were Caucasian from USA.
The group of cases included 289 subjects with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 50 [32;74] years.
The group of healthy volunteers included 289 individuals with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 48.7 [36;75] years.
Table 1: Description and stratification study of the different collections
'Clinical forms :
PP: Primary Progressive
RP : Relapsing Progressing (or SP : Secondary Progressive)
RR: Relapsing Remitting All the Fst values found for each collection indicate that these samples are genetically homogeneous. They can be therefore used in association analysis.
1.2. DNA extraction:
Genomic DNA was extracted from EDTA anticoagulated peripheral blood according to a Standard proteinase K digestion and a modified salting out extraction method of Miller and co-workers (1988).
2.1 GENOTYPING
2.1.1 Methods for stratification analyses: Beckman UHT protocol:
Assay Design
Design of the two PCR primers and one SNP-IT primer for each marker set was performed using Autoprimer.com (http://www.autoprimer.com). The Autoprimer.com design engine reads each sequence and designs three primers; forward and reverse PCR primers and a SNP-IT primer for the single base extension step. Once primers are picked for each sequence, they are then assembled into groups of 12 by SNP extension type (e.g., A/G, T/C).
Each group, or panel of 12 markers, must be of the same extension type for processing on the UHT since each extension mix contains two labeled terminators (Bodipy-Fluorescein and TAMRA). Each group of twelve is referred to as a panel of markers. Autoprimer.com automatically optimizes the grouping of the markers by extension mix and appends tag sequences to the 5' ends of the SNP-IT primers, which are complementary to the tags immobilized on the microarray plate.
PCR
A five-micro liter PCR was performed in 384-well plates (MJ Research, Watertown, MA, USA) using 75-uM dNTPs and 0.5U AmpliTaq® Gold (Applied Biosystems) in IXPCR buffer. Two nanograms of genomic DNA were used in each reaction.
The 24 PCR primers were pooled and added such that each was at a final concentration of 50 nM. Thermal cycling was performed in DNA Engine Tetrad thermal cyclers (MJ Research) using the following program: 95°C for 5 seconds followed by 45 cycles of 95°C for 30 seconds; 50°-55°C for 55 seconds; 72°C for 30 seconds. The first six cycles used an annealing temperature of 50°C after which the annealing temperature was increased by 0.2°C in the subsequent cycles until the annealing temperature reached 55°C. After the last cycle, the reaction was held at 72°C for 7 minutes followed by a 4°C hold.
PCR Clean-Up
Following PCR, 384-well plates were centrifuged briefly to collect the contents and 3 uL of a cocktail containing 0.67 U exonuclease I (USB, Cleveland, OH, USA) and 0.33 U shrimp alkaline phosphatase (SAP; USB) was added. Sealed plates were incubated for 30 minutes at 37°C to degrade residual PCR primers and dNTPs, and 10 minutes at 100°C to inactivate the enzymes.
SNP-IT Reaction
To the ExoI/SAP-treated PCR, we added 7 uL of a cocktail containing one TAMRA- labeled and one Bodipy-Fluorescein-labeled nucleotide terminator (PE-NEN, Boston, MA, and Molecular Probes, Eugene, OR, USA), the two remaining unlabeled terminators, 26.6 mM MgC12, 266 mM Tris-HCl pH 9.5, two allele-specific self-extension control primers, and a thermostable, 3'exonuclease-deficient polymerase such as Thermo Sequenase (Amersham Biosciences, Piscataway, NJ, USA). The total reaction volume was 15 uL. Plates were re-sealed and thermal cycled using the following program: 96°C for 3 minutes followed by 45 cycles of 94°C for 20 seconds; 40°C for 11 seconds. After the last cycle, the reaction was held at 4°C.
Hybridization and Washing
Following SNP-IT extension, 8 μL of hybridization buffer (5M NaCl, 0.5 M EDTA, 580 mM morpholinoethane sulphonic acid (MES) pH 6.6, IX Denhardt's Solution) was added and a portion of the mixture was applied to the well of a UHT microarray plate. Plates were incubated in a humidified container at 42°C for 2 hours to promote hybridization of the SNP-IT primers to their complementary immobilized tags. Plates were rinsed with UHT wash buffer using a conventional plate washer to remove unhybridized material and were then ready for imaging. SNPstream UHT Array Imager
The SNPstream Array Imager is based upon a two-laser, two-color approach. Each sample is illuminated with a 488-nm laser beam and subsequently with a 532-nm laser beam to excite the fluorescent oligonucleotides captured on the UHT microarray plates. The system 5 contains two emission band filters. Fluorescence emission from 488-nm excitation (Bodipy- Fluorescein) is captured in a band 50 nm wide, centered at 535nm. Fluorescence emission from 532-nm excitation (TAMRA) is captured in a band 55 nm wide, centered at 590 nm. A colorcorrected custom lens, of high numerical aperture and 100-um A 2 X3 well area is imaged per frame. Sixty- four 2 X3 well images/color are taken per plate for a 10 total of 384 wells. Total time required for the process is approximately seven minutes/plate.
Data Analysis
Generation of genotype calls from spots detected using the SNPstream UHT Array Imager involves two discrete steps. First, the location and intensity of a spot within the well and 15 plate is determined for each wavelength; second, a genotype call is made based on the relative fluorescent intensities of each spot. Once a genotype call has been made, results are written to an Oracle® database where the data can easily be retrieved for viewing.
Spot detection is an automatic process performed by UHTImage software. Positive controls in each well are used to align the grids around the 4 X4 element array. Once a grid 20 is drawn, each spot is analyzed for morphology (i.e., circular shape and regular pixel intensity across each spot). Spots with low intensity or unusual morphology are marked as empty or fail. For each spot that passes the morphology test, an intensity value is generated and loaded into the UHT database. Failed spots are carried through the analysis but are flagged for the user to review.
25 Genotype calling is performed once all spot intensities are in the database for each sample within a plate. Each SNP marker is analyzed separately using UHT GetGenossoftware. This software automatically creates genotype calls based on the intensity value of each spot at each wavelength for a given sample. These calls are based on how the sample points cluster when plotted on a X, Y graph where X corresponds to the intensity in the
30 488-nm channel and Y to that of the 532-nm channel. If a point falls between clusters or the intensity of the point is too low, the sample is failed. Otherwise the point is called as XX, XY, or YY with the X's and Y's being replaced by the actual allele calls (A5C5G5T). UHT GetGenos uses a proprietary algorithm to determine the clusters and the genotypes for each sample. After the genotype calling, the results are stored in the database by microarray plate number, well, and spot location.
2.1.2 Methods for Whole Genome analysis: Affymetrix method:
Use of Affymetrix genotyping technology have been described in Kennedy, G.C., et al. Nature Biotechnology 21, 1233-1237, 2003 (Large-scale genotyping of complex DNA). ; Liu, W.M., et al. Bioinformatics 19, 2397-2403, 2003. (Algorithms for Large Scale Genotyping Microarrays) ;Matsuzaki, H., et al. Genome Research 3, 414-25, 2004. (Parallel Genotyping of over 10,000 SNPs using a One Primer Assay on a High Density Oligonucleotide Array) ;Paez, J.G., et al. Nucleic Acids Research 32(9), 2004. (Genome coverage and sequence fidelity of Φ29 polymerase-based multiple strand displacement whole genome amplification); Matsuzaki, H., et al.. Nature Methods, 1, 109-111, 2004 (Genotyping over 100,000 SNPs on a Pair of Oligonucleotide Arrays); Huang, J., et al., M.H. Human Genomics 1(4), 287-99, 2004. (Whole Genome DNA Copy Number Changes Identified by High Density Oligonucleotide Arrays); Mitra N, et al. Cancer Research 64 (21), 8116-25, 2004 (Localization of cancer susceptibility genes by genome-wide single- nucleotide polymorphism linkage-disequilibrium mapping), all of which are herewith incorporated by reference.
DNA preparation:
For each individual assayed, 250 ng of genomic DNA are digested separately with 10 U of Xbal or HmdIII (New England BioLabs) in volumes of 20 μL for 2 hours at 37 °C. Following heat inactivation at 70 °C for 20 minutes, 0.25 μM of Xbal adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -CTA GAG ATC AGG CGT CTG TCG TGC TCA TAA- 3') (Affymetrix), or HmdIII adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -AGC TAG ATC AGG CGT CTG TCG TGC TCA TAA-3') (Affymetrix) are ligated to the digested DNAs with T4 DNA Ligase (New England BioLabs) in 25 μL for 2 hours at 16 °C. The ligations are stopped by heating to 70 °C for 20 minutes, and then diluted 4- fold with water. For each ligation reaction, two to three PCRs are run in order to generate > 40 μg of PCR products. Each PCR contains 10 μL of the diluted ligation reactions (25 ng of starting DNA) in 100 μL volumes containing 1.0 μM of primer (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3'), 0.30 mM dNTPs, 1.0 mM MgSO4, 5 U Platinum® Pfa Polymerase (Invitrogen), PCR Enhancer (Invitrogen) and Pfa Amplification Buffer (Invitrogen). 30 cycles of PCRs are run with the following cycling program: 94 °C denaturation for 15 seconds, 60 °C annealing for 30 seconds, and 68 °C extension for 60 seconds. As a check, 3 μL of PCR products are visualized on 2% TBE agarose gels to confirm the size range of amplicons. The PCR products are purified over MinElute 96 UF PCR Purification plates (Qiagen), and recovered in 40 μL of EB buffer (Qiagen). PCR yields are measured by absorbance readings at 260 nm, and adjusted to a concentration of 40 μg per 45 μl. To allow efficient hybridization to 25-mer oligonucleotide probes, the PCR products are fragmented to < 100 bp with DNAse I. 0.20 U of DNAse I (Affymetrix) is added to 40 ug of purified PCR amplicons in a 55 μL volume containing Fragmentation Buffer (Affymetrix) for 35 minutes at 37 °C, followed by heat inactivation at 95 °C for 15 minutes. Fragmentation products are visualized on 4% TBE agarose gels. The 3' ends of the fragmented amplicons are biotinlyated by adding 214 μM of a proprietary DNA labeling reagent (Affymetrix) using Terminal Deoxynucleotidyl Transferase (Affymetrix) in 70 μL volumes for 2 hours at 37 °C, followed by heat inactivation at 95 °C for 15 minutes.
Allele Specific Hybridization to Oligonucleotide Arrays
The fragmented and biotinylated PCR amplicons are combined with 11.5 μg/mL human Cot-1 (Invitrogen) and 115 μg/mL herring sperm (Promega) DNAs. The DNAs are added to a hybridization solution containing 2.69 M tetramethylamonium chloride (TMACl), 5.77 mM EDTA, 56 mM MES, 5 % DMSO, 2.5 X Denhardt's solution, and 0.0115% Tween-20 in a final volume of 260 μL. The hybridization solution was heated to 95 °C for 10 minutes then placed on ice. After warming to 48 °C for 2 minutes, 200 μL of the hybridization solution is injected into cartridges housing the oligonucleotide arrays (Affymetrix GeneChip® IOOK Mapping Set: 50K Array Xba 240 and 50K Array Genotyping over 100,000 SNPs Hind 240). Hybridizations are carried out at 48 °C for 16 to 18 hours in a rotisserie rotating at 60 rpm. Following the overnight hybridization, the arrays are washed with 6X SSPE and 0.01% Tween-20 at 25 °C, then more stringently washed with 0.6X SSPE and 0.01% Tween-20 at 45 °C. Hybridization signals are generated in a three step signal amplification process: lOμg/mL streptavidin R-phycoerythrin (SAPE) conjugate (Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 °C; followed by the addition of 5μg/mL biotinylated goat anti-streptavidin (Vector) to increase the effective number of biotin molecules on the target; and finally SAPE is added once again and washed extensively with 6X SSPE and 0.01% Tween-20 at 30 °C. The SAPE and antibody were added to arrays in 6X SSPE, IX Denhardt's solution and 0.01% Tween-20 at 25 °C for 10 minutes each. Following the final wash, the arrays are kept in Holding buffer (10OmM MES, IM [Na+], 0.01% Tween-20). The washing and staining procedures are run on Affymetrix fluidics stations. Arrays are scanned using GC S3000 scanners with AutoLoaders (Affymetrix). Scan images are processed to get hybridization signal intensity values using GCOS 2.0 software (Affymetrix). The DM genotype calling algorithm is implemented in GenoTyping Tools (GTT) (Affymetrix) and GDAS 3.0 (Affymetrix) analysis software.
2.2 STATISTICAL ANALYSIS
Design: We have decided to analyze 2 different populations in parallel to minimize the risk of type I errors (false positives) due to the relatively limited sample size. In our case, the 2 populations have the same euristic value and neither one represents an exploratory or a confirmation sample. Rather they represent 2 complementary views of the same analytical problem and only positive results that are cross-confirmed are retained as valid. The following paragraphs detail the statistics that we applied to perform our analyses. A third population (SeraCare) was studied to further confirm the results.
PART A: Descriptive statistics
2.2.1 Genetic homogeneity: FST test and Pritchard and Rosenberg test
A stratification effect is a non-homogeneous representation of populations between the case and the control groups due to genetic heterogeneity, which may lead to spurious association results and replication problems.
If cases and controls contain an admixture of different groups (for example, based on ethnicity), we expect to find a consistent pattern of allele-frequency differences between cases and controls, at many random loci throughout the genome, this difference exceeding the significant p-value for association at more than 5% of these random loci.
The power to detect stratification will depend on the number of loci used to test for homogeneity. Consequently, we have chosen a large number of unlinked SNP markers (n=86) [The complete list is in Annex I]. These SNPs have been selected under the following conditions:
1 Minor allele frequency > 30% (highly polymorph)
2 Inter SNP distance > 10Mb (genetically independent)
3 Location in each chromosome (genome wide scale) but not in a known associated region for the studied disease (not associated with the disease)
All cases and controls were genotyped for all the unlinked genetic markers set using the Beckman technology.
Two methods testing for genetic heterogeneity have been implemented in Serono Genetics Institute:
1. Fst test (Wright 1951) is an ANOVA-based method. The Fst value quantifies the loss of heterozygosity due to existence of a hierarchical structure. If it is different from 0, it means that the population under study are genetically heterogeneous, since allelic frequencies are different between populations. 2. Pritchard & Rosenberg test (Am. J.Hum. Genet. 65:220-228, 1999) calculates an overall chi-square statistic of allelic frequency differences between cases and controls.
If the Fst and the Pritchard & Rosenberg tests do not show statistically significant results (p-value > 5%), cases and controls are considered homogenous and can be used for case- control association study.
However, statistically significant results at these tests do not necessarily mean that these populations must be discarded. Further analyses can assign each subject to a specific subpopulation and identify outliers (Structure software, Pritchard, 2002), that can be removed in order to restore homogeneity.
When the admixture is such that we can not identify clear subpopulations, we can adopt another approach, termed Genomic Control {Devlin and Roeder 1999): given that in the presence of population substructure, the standard chi-square statistic is inflated by a multiplicative factor, which is proportional to the degree of stratification, we can estimate and incorporate this multiplicative factor (lambda) into the disease - marker association tests (by rescaling the chi-square statistic) to correct for background population differences.
PART B: Inferential statistics
2.2.2 Univariate analysis
I. Hardy- Weinberg Equilibrium / Disequilibrium [HWE/D] : significance in Cases and in Controls
The Hardy- Weinberg law regulating equilibrium (HWE) is the central theory of population genetics, explaining why populations have a stable genetic pattern across generations and is based on four assumptions:
1. Populations are panmict (couples are formed at random) and their gametes meet randomly;
2. Populations are "Infinite" (large population size to minimize sampling variations);
3. There are no selection, mutation, migration (=no allele loss or allele gain);
4. Generations are discrete (no mating between different generations).
According to these hypotheses, the control population used in case-control association studies must respect this equilibrium, if sampled randomly. On the contrary, the population of cases can present some disequilibrium that may point to "mutations" underlying the disease, since cases are not a random representation of the general population.
Accordingly, we tested HWE for each SNP in the control population, and we removed from the study each SNP presenting a deviation from the equilibrium. In fact, any such deviation might be due to several different reasons, but especially to technical issues (e.g. neighbouring SNPs causing imbalance of the polymerase chain reaction products or affecting the genotyping assay). HWE test therefore serves two objectives: data review and quality check as well as detection of possible mutation. The test described by Weir in Genetic Data Analysis II (Sinauer, 1996) has been implemented using a chi-square statistics (ldf). The SNPs with results showing significant deviation from HWE (pvalue < 0.02) were considered in disequilibrium and were not validated, a positive deviation demonstrating an excess of homozygotes (or lack of heterozygotes) and a negative deviation being due to an excess of heterozygotes (or lack of homozygotes).
Hardy- Weinberg equilibrium statistics were calculated separately for cases and controls data and Observed and Expected genotype frequencies were compared using a Pearson's χ2 test. A departure from Hardy- Weinberg equilibrium (HWE) in case population may indicate that a mutation had occurred, which could be responsible for increasing the risk for the disease.
II. Tests on allelic frequencies, genotypic frequencies, HWD
In the univariate analysis (or Single Point Analysis), SNPs were analysed one by one. The Pearson's 2x2 χ2 test was used to compare allele frequencies between cases and controls, while we used a 3x2 χ2 test for the overall difference in genotype frequencies. The Exact Fisher test was performed wherever the minor expected frequency for each cell of the χ2 table is < 5.
Additional statistics include (i) the difference between allelic frequencies in cases and in controls (the larger the difference in allelic frequency for a given SNP, the more probable is an association between the genomic region containing that SNP and the disorder), (ii) the Odds Ratio (OR) of the association and (iii) the population Attributable Risk (pAR). The "chosen" allele is the allele for which the frequency is increased in cases compared to controls.
We considered a p-value = or < 0.05 as threshold to consider the tests as significant for screening, with the only exception relative to HW test where the threshold is = or < 0.02.
III. Mantel Haenszel test: comparison of the significant findings across the 2 populations.
The relationships between genetic susceptibility to MS and allele frequencies have been studied for many markers (N = 95 938) in at least one of the two populations (Rennes & Huddinge). Data from most of these SNPs (N=82 925) are available for the two populations (Rennes & Huddinge): therefore, they represent the basis to evaluate associations that are observed in the two populations simultaneously.
We used the Mantel-Haenszel χ2 test which was designed for case-control studies in which the effect of an exposure-factor (Allele) on the outcome (MS) is investigated according to a stratification factor (Population).
A program was written at SGI to perform the Mantel-Haenszel test using data from n independent populations (Principles of Biostatistics, Second Edition, Marcello Pagano & Kimberlee Gauvreau, Duxbury-Thomson Learning).
2.2.3. Odds Ratio (OR)
By estimating the allelic Odds Ratio (OR) we evaluate the probability of having the disease when carrying a given allele (= chosen [or 'risk'] allele) compared to not carrying it. An OR higher than 1 shows that the probability of having multiple sclerosis is higher when carrying the 'risk' allele [or genotype or haplotype] than when carrying the other ones. The genotypic OR allows the identification of the 'risk' genotype(s) for an associated biallelic marker. The genotypic odds ratio was calculated and Tables 2 to 5 shows the significant results of the respective genes analysed.

Claims

L A method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence of a susceptibility alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
2. A method of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder, the method comprising detecting the presence of a susceptibility alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
3. The method according to claim 1 or 2, wherein said susceptibility alteration is a single nucleotide mutation.
4. The method according to any one of claims 1 to 3, wherein said susceptibility alteration is located within the 3' or 5' region of the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
5. The method of any one of claims 1 to 3, wherein the susceptibility marker is selected from markers as listed in Table 2a for AUTS2, Table 3a for PARK2, Table 4a for PSENl and/or Table 5a for KCNIP4.
6. The method of any one of claims 1 to 3, wherein the susceptibility marker is selected from markers as listed in Table 2b for AUTS2, Table 3b for PARK2, Table 4b for PSENl and/or Table 5b for KCNIP4.
7. The method according to any one of claims 1 to 6, wherein the presence of an alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene is detected by sequencing, selective hybridisation and/or selective amplification.
8. The use of a functional AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide or a nucleic acid encoding the same, for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
5 9. The use of a AUTS2, PARK2, PSENl and/or KCNIP4 agonist for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
10. The use of claim 9, wherein the agonist is a natural ligand of AUTS2, PARK2, PSENl 10 and/or KCNIP4.
11. The use of claim 9, wherein the agonist is an antibody that selectively binds AUTS2, PARK2, PSENl and/or KCNIP4.
15 12. The use of any one of claim 8 to 11 for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject presenting a susceptibility alteration in the AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide.
20 13. The use of claim 12, wherein said susceptibility alteration is a marker selected from markers as listed in Table 2a or 2b for AUTS2, Table 3a or 3b for PARK2, Table 4a or 4b for PSENl and/or Table 5a or 5b for KCNIP4.
14. The use of a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide as a target for 25 screening candidate drugs for treating or preventing multiple sclerosis or a related disorder.
15. A method of selecting biologically active compounds, said method comprising contacting a candidate compound with a AUTS2, PARK2, PSENl and/or KCNIP4 gene or polypeptide and selecting compounds that bind said gene or polypeptide.
30
16. A method of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide with a candidate compound, and selecting compounds that bind said AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide at the surface of said cells and/or that modulate the activity of said AUTS2, PARK2, PSENl and/or KCNIP4 polypeptide.
17. The method of claim 15 or 16, further comprising a step of assaying the activity of the selected compounds in a model of multiple sclerosis or a related disorder.
18. A method of treating multiple sclerosis or a related disorder in a subject in need of such treatment comprising of administering to the subject a compound that modulates the synthesis, expression or activity of one or more of the genes or gene products of the genes listed in Table 2a or 2b for AUTS2, Table 3a or 3b for PARK2, Table 4a or 4b for PSENl and/or Table 5a or 5b for KCNIP4 in a therapeutically effective amount so that at least one symptom of the multiple sclerosis or a related disorder is ameliorated.
19. A method of treating multiple sclerosis or a related disorder in a subject in need of such treatment and wherein the subject has a susceptibility alteration in a AUTS2, PARK2, PSENl and/or KCNIP4 gene comprising of administering to the subject a therapeutically effective amount of a medication for MS.
20. The method of claim 19, wherein medication for MS is interferon-beta, preferably interferon-beta Ia.
21. The method of claim 19 or 20 wherein the susceptibility alteration is selected from one or more of the susceptibility markers listed in Table 2a or 2b for AUTS2, Table 3 a or 3b for PARK2, Table 4a or 4b for PSENl and/or Table 5a or 5b for KCNIP4.
22. The method of claims 19 to 21 wherein said susceptibility alteration is located within the 3' or 5' region of the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
23. The method of claims 19 to 22 wherein said susceptibility alteration is a single nucleotide mutation.
24. A kit for the identification of a genetic polymorphism pattern at the AUTS2, PARK2, PSENl and/or KCNIP4 gene associated with increased risk of the presence of or predisposition to multiple sclerosis or a related disorder in a subject, said kit comprising: (a) DNA sample collecting means, and (b) means for determining a genetic polymorphism pattern for the AUTS2, PARK2, PSENl and/or KCNIP4 gene.
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