+

WO2013179061A1 - Tomm40 en tant que marqueur pour la maladie de parkinson - Google Patents

Tomm40 en tant que marqueur pour la maladie de parkinson Download PDF

Info

Publication number
WO2013179061A1
WO2013179061A1 PCT/GB2013/051463 GB2013051463W WO2013179061A1 WO 2013179061 A1 WO2013179061 A1 WO 2013179061A1 GB 2013051463 W GB2013051463 W GB 2013051463W WO 2013179061 A1 WO2013179061 A1 WO 2013179061A1
Authority
WO
WIPO (PCT)
Prior art keywords
optionally
subject
disease
parkinson
genotype
Prior art date
Application number
PCT/GB2013/051463
Other languages
English (en)
Inventor
Lefkos MIDDLETON
Lachlan COIN
Federico CALBOLI
Original Assignee
Imperial Innovations Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imperial Innovations Ltd filed Critical Imperial Innovations Ltd
Publication of WO2013179061A1 publication Critical patent/WO2013179061A1/fr

Links

Classifications

    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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

Definitions

  • the present invention relates to assessment and treatment relating to Parkinson's Disease and motor Parkinsonisian features.
  • Old age dementias are chronic and relentlessly progressive neurodegenerative disorders which are set to become the world's largest socioeconomic healthcare problem, as life expectancy continues to increase.
  • the number of dementia patients world-wide was estimated at 24 million in 2001 and is predicted to increase to 42 million in 2020 and may reach 80 million sufferers by 2040 (Ferri et al, 2005, Comas-Herrera et al, 2005)
  • Parkinson's disease (PD) is the second most common progressive neurodegenerative disease of ageing after Alzheimer's disease (AD), albeit considerably less prevalent.
  • the key neuropathological substrate of PD and the two PD- associated forms of dementia are a selective neuronal loss of dopaminergic neurons in the substantia nigra and widespread intraneuronal inclusions of aggregated a-synuclein, known as Lewy bodies (LB) and Lewy neuritis (McKeith et al, 1996, Gibb and Lees, 1988, Spillantini et al, 997).
  • DLB and PDD share common neuropsychological features of "subcortical” impairment with prominent attentional and planning (dysexecutive) deficits associated with hallucinations, fluctuating confusion and behavioral abnormalities (McKeith et al, 2005, Emre et al, 2007)
  • the timing of the onset of dementia in relation to the onset of motor manifestations is the only criterion differentiating DLB from PDD.
  • DLB is the second most common age related neurodegenerative dementia 3 , with a reported prevalence of more than 20% of all dementia cases (Mc Keith et al, 1996, Zaccai et al, 2005) .
  • PDD accounts for 3-4% of all dementia cases (Aarsland et al, 2001, De lau et al, 2005) .
  • PD microtubule-associated protein tau
  • MTT microtubule-associated protein tau
  • APOE maps to chromosome 19 and is in linkage disequilibrium with the Translocase of Outer Mitochondrial Membrane 40 homolog (TOMM40) gene.
  • TOMM40 Translocase of Outer Mitochondrial Membrane 40 homolog
  • rs10524523 has been identified within the intervening sequence of intron 6 (IVS6) and, through a combined deep sequencing and phylogenetic approach, was shown to be strongly associated with the age of onset of dementia in Alzheimer's disease (Lutz et al, 2010, Roses et al, 2010).
  • the very long (VL) IVS6 poly T repeat was associated with increased risk and lower age of onset, independent of the risk imparted by the APOE ⁇ 4 allele.
  • a first aspect of the invention provides a method for aiding in determining likelihood of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD); or for categorising or determining prognosis, optionally a relatively high or relatively low likelihood of developing or worsening dementia, for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features; and/or in selecting a therapeutic strategy for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features, the method comprising the step of assessing the subject's genotype for TOMM40, optionally in intron 6 (IVS6) of the TOM 40 gene, optionally at position rs10524523.
  • the subject may, for example, be a subject considered to have at least early motor Parkinsonisian features, for example early signs of motor symptoms such as tremor, rigidity and bradykinesia.
  • the aiding in categorising or determining prognosis may be aiding in determining whether the subject has a relatively high or relatively low likelihood of developing or worsening dementia associated with Parkinson's Disease or motor Parkinsonisian features.
  • two PD- associated forms of dementia are generally termed Dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).
  • DLB Dementia with Lewy bodies
  • PPD Parkinson's disease dementia
  • the timing of the onset of dementia in relation to the onset of motor manifestations is the only criterion differentiating DLB from PDD.
  • Dementia occurring two or more years after onset of motor symptoms may be defined as PDD, as discussed in Example 1.
  • the method of the invention may, for example, be useful for aiding in determining whether the subject has a relatively high or relatively low likelihood of developing Dementia with Lewy bodies (DLB) ie dementia occurring within a one or two year period of onset of motor symptoms of Parkinson's Disease or motor Parkinsonisian features.
  • DLB Lewy bodies
  • Parkinson's Disease and “motor Parkinsonisian features” will be well known to those skilled in the art.
  • a clinical diagnosis of Parkinson's Disease may be made as well know to those skilled in the art.
  • As defined by the Queen Square brain bank clinical diagnostic criteria (and for example, as set out in Example 1), such a diagnosis may be made when the subject has bradykinesia and at least one of the following: muscular rigidity, rest tremor, postural instability with asymmetrical onset and good response to levodopa in the initial phases of the disease as validating evidence (Gibb and Lees, 1989). See, for example, Nelson, P.T., Kryscio, R.J., Jicha, G.A. & Abner, E.L.
  • DLB or PDD DLB or PDD
  • MMSE MMSE-proven dementia with Lewy bodies.
  • a diagnosis of DLB or PDD may be made as well known to those skilled in the art, for example if, in addition to a diagnosis of Parkinson's Disease or motor Parkinsonisian features, the presence of dementia is identified, for example as set out in Example 1 , "Clinical assessment" section.
  • neuropsychological features of subcortical impairment may be present, with prominent attentional and planning (dysexecutive) deficits associated with hallucinations, fluctuating confusion and behavioural abnormalities (Emre et al, 2007), as noted above.
  • the method for aiding in determining likelihood of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD) may be applied to any subject and may, for example, be applied to a subject who may have other recognised risk factors for Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD) , for example a subject who is known to have a genotype (other than at TOMM40) linked with Parkinson's Disease, at least in a particular population, for example an LRRK2 genotype linked with Parkinson's Disease, for example LRRK2 G2019S, as known to those skilled in the art (Hulihan et al, Lancet Neurology, 2008).
  • the LRRK2 G2019S genotype has a known penetrance of ⁇ 80% by age of 80 i.e. 80% of those with the mutation have PD by age of 80 (Hulihan et al, Lancet Neurology, 2008).
  • Other risk factors may include one or two or three or more of head trauma, male gender, age (for example over 60), diabetes, one or more of a number of genes for familial PD (a total of around 16 have been identified), hypertension, rural living, exposure to pesticides or heavy metals.
  • the method for categorising or determining prognosis, optionally a relatively high or relatively low likelihood of developing or worsening dementia, for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features may typically be applied with a subject who has been diagnosed with PD/ motor Parkinsonisian features but not DLB or PDD. However, it may also be useful with a subject who has been diagnosed with PD/ motor Parkinsonisian features and presents with early symptoms of hallucinations, cognitive decline or dysexecutive syndrome suggesting possibly an early stage of DLB or PDD
  • the subject typically is a human subject.
  • a clinician may wish to take into account the subject's genotype at TO M40, as discussed herein, alongside other parameters as noted above in arriving at a diagnosis of PD/ motor Parkinsonisian features or DLB or PDD. Accordingly, assessing a subject's genotype at TO M40, as discussed herein, may be useful in aiding in a diagnosis of PD/ motor Parkinsonisian features or DLB or PDD.
  • a subject may be considered otherwise at risk of Parkinson's Disease or motor Parkinsonisian features from a combination of known risk factors, such as one or two or three or more of: a number of genes for familial PD (a total of 16, for example an LRRK2 genotype linked with Parkinson's Disease, as discussed above and well known to those skilled in the art); age; male gender; diabetes; hypertension; rural living; exposure to pesticides, heavy metals etc; and head trauma. (Wirdefeldt et al, 2011).
  • the subject's genotype may be determined at the time of considering a diagnosis of PD/motor Parkinsonisian features, or risk thereof; or it may be (or have been) determined separately, for example as part of a wider genetic characterisation of the subject.
  • the subject's characterisation may then be stored; and accessed when considering a diagnosis of PD/ motor Parkinsonisian features, or risk thereof, for the subject.
  • the genotyping may be done on any suitable tissue from the subject, for example on a blood sample from the subject, as will be well known to those skilled in the art.
  • the genotyping may be done by any suitable technique for determining genotype, as will be well known to those skilled in the art and as discussed further below.
  • TOMM40 will be well known to those skilled in the art. TOMM40 sequences are well known and a human genomic sequence may be found at, for example,
  • Nucleotides 1 to 2908 relates to TOMM40
  • intron 6 (I S6) of the TOMM40 gene and position rs10524523 are also well known to those skilled in the art and are discussed in, for example, Lutz et al (2010) Alzheimers Dement 6(2), 125-131 (see for example Figure 3: diagram of exons 6(E6) to 10 (E10); and Linnertz et al (2012) PLoS ONE, 7(2), e30994; and in Example 1.
  • Position rs10524523 is considered to be the site of a deletion/insertion polymorphism, particularly a poly-T deletion/insertion polymorphism in which the number of T residues varies between alleles.
  • Figure 1 shows a distribution of poly-T repeat lengths at this position. See also Linnertz et al (2012), supra.
  • NCBI snp database shows rs 10524523 as
  • VL Very long
  • the method of the invention may comprise the step of determining whether the subject's genotype for TOMM40, optionally in intron 6, optionally at rs10524523, is heterozygous, optionally determining whether there is a difference in the length of a poly-T stretch at rs10524523 between the patient's alleles, optionally whether the subject's genotype at rs10524523 is S VL or whether the difference in the length of the poly-T stretch (at rs10524523) is more than or equal to 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 base pairs, optionally more than or equal to 12, 13, 14, 15, 16, 17 or 18 base pairs, for example more than or equal to 15 base pairs.
  • TOMM40 heterozygosity for example in intron 6, for example at rs10524523, for example genotype S/VL at this position or a difference of roughly around 15 base pairs between the poly-T stretches at this position, may affect the way in which the TOMM40 gene is expressed (for example transcribed, processed and/or translated), which may also affect the way in which the complex containing the TO M40 polypeptide is assembled
  • the skilled person will be well aware of techniques and reagents suitable for and useful in assessing genotype for TOMM40, optionally in intron 6, optionally at rs 0524523, for example for assessing whether the genotype is heterozygous or determining whether there is a difference in the length of a poly-T stretch at rs10524523 between the subject's alleles, optionally whether the subject's genotype at rs10524523 is S/VL or whether the difference in the length of the poly-T stretch (at rs10524523) is
  • PCR polymerase chain reaction
  • PCR- RFLP PCR restriction fragment length polymorphism
  • a PCR based technique may be used, for example as described in Example 1 or in Linnertz et al (2012).
  • suitable techniques are described in Example 1 , in Lutz et al (2010) supra or in Linnertz et al (2012), supra, for example.
  • suitable primer sequences for analysis at position rs10524523 are given in Example 1 and in Linnertz et al (2012), for example
  • Length of poly-T stretches may be determined by, for example, sequencing techniques or by length determination, for example using capillary electrophoresis. Techniques other than or in addition to PCR may also be used, for example ligase based detection techniques such as the Ligase Detection Reaction (LDR), as well known to those skilled in the art. Other examples of suitable techniques include QB replicase and NASBA (nucleic acid sequence based amplification), also called 3SR, for example as described in Compton (1991) Nature 350, 91-92 and AIDS (1993), Vol 7 (Suppl 2), S108 or SDA (strand displacement amplification), for example as described in Walker et al (1992) Nucl. Acids Res. 20, 1691-1696. The polymerase chain reaction is particularly preferred because of its simplicity.
  • LDR Ligase Detection Reaction
  • the genotype for TO 40 is determined on a sample obtained from the subject. Any sample that contains TO M40 nucleic acid, typically genomic DNA, in sufficient quantity to be assessed by the chosen method may be used. For example, a blood, urine, hair, skin or other epithelial cell sample may be used, as well known to those skilled in the art.
  • the techniques for assessing TOMM40 genotype, optionally in intron 6, optionally at rs 0524523, may make use of one or more nucleic acids capable of hybridising to the TOMM40 nucleic acid, typically genomic DNA.
  • the hybridising nucleic acid(s) which is (are) used in the methods of the invention may further comprise a detectable label, as will be well known to those skilled in the art.
  • detecttable label any convenient radioactive label such as 32 P, 33 P or 35 S which can readily be incorporated into a nucleic acid molecule using well known methods; any convenient fluorescent or chemi!uminescent label which can readily be incorporated into a nucleic acid is also included.
  • detecttable label also includes a moiety which can be detected by virtue of binding to another moiety (such as biotin which can be detected by binding to streptavidin); and a moiety, such as an enzyme, which can be detected by virtue of its ability to convert a colourless compound into a coloured compound, or vice versa (for example, alkaline phosphatase can convert colourless o-nitrophenylphosphate into coloured o-nitrophenol).
  • the nucleic acid probe may occupy a certain position in a fixed assay and whether the nucleic acid hybridises to the said TOMM40 nucleic acid can be determined by reference to the position of hybridisation in the fixed assay.
  • the detectable label may also be a fiuorophore-quencher pair as described in Tyagi & Kramer (1996) Nature Biotechnology 14, 303-308.
  • the nucleic acid may be branched nucleic acid (see Urdea ef at (1991) Nuc!. Acids Symposium Series 24, 197-200).
  • the method of the invention need not include a step of assessing the APOE genotype of the subject. Whilst the present inventors have identified that the APOE genotype ⁇ 4/ ⁇ 4 may be linked to a marginally significant earlier onset of dementia (see table 5 of Example 1) it is not considered that consideration of APOE genotype alongside TOM 40 genotype provides useful further information on the likelihood of developing PDD or DLB, or developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD); or for categorising or determining prognosis, optionally a relatively high or relatively low likelihood of developing or worsening dementia, for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features. Thus, in an embodiment, the method does not include the step of assessing the APOE genotype of the subject.
  • the subject may be considered to be at higher risk of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD); disease progression, for example at higher risk of developing Dementia with Lewy Bodies (DLB) or Parkinson's Disease Dementia (PDD), for example of developing DLB (typically within a one or two year period of onset of motor symptoms of Parkinson's Disease or motor Parkinsonisian feature,) if the subject's TOMM40 genotype, for example in intron 6, for example at rs10524523, is heterozygous, for example when there is a difference in the length of a poly-T stretch at rs10524523 between the subject's alleles, for example when the subject's genotype at rs10524523 is S VL or wherein the difference in the length of the poly-T stretch is more than or equal to 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 base pairs, for example more than or
  • determining the TOM 40 genotype in the sample may in itself allow categorising or determining prognosis in a subject with PD/motor Parkinsonisian features (or otherwise at risk thereof), or selection of a therapeutic strategy for a subject with PD/ motor Parkinsonisian features (or at risk thereof); or more typically it may be used by the clinician as an aid in categorising or determining prognosis or selection of a therapeutic strategy.
  • the clinician may take into account the number and severity of the various PD/ motor Parkinsonisian feature symptoms (or risk factors) and/or DLB or PDD symptoms known to those skilled in the art, for example hallucinations or mild cognitive symptoms, as mentioned herein. It will be appreciated that the clinician will wish to take in to account these or other factors, as well as consider the TOMM40 genotype, as set out above, before categorising or determining prognosis or selection of a therapeutic strategy.
  • the method of the invention may further comprise the step of selecting a treatment regime making use of the information on the subject's TOMM40 genotype. Determination of the subject's TOMM40 genotype, as set out above, will be useful to the clinician in determining how to manage (or potentially prevent or delay) the PD/ motor Parkinsonisian features in the subject. For example, since the heterozygous genotype, as indicated above, is considered to be associated with a higher risk of developing dementia, the clinician may use the information concerning the TO M40 genotype to facilitate decision making regarding treatment of the subject. Thus, if the TOMM40 genotype is indicative of a lower probability of developing dementia unnecessary treatments may be avoided.
  • the TO 40 genotype is indicative of a higher probability of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD), for example of developing or worsening dementia
  • therapy directed to disease modification and/or to dementia may be the preferred treatment, particularly if motor Parkinsonisian features or early signs of dementia are present.
  • determining whether the TO M40 genotype is indicative of a higher probability of developing or worsening dementia may help the clinician to decide or advise on appropriate counseling or advice to the subject and/or family.
  • Treatment directed to disease modification and/or to dementia and/or more frequent monitoring may be chosen if a subject has a TOMM40 genotype indicative of a higher probability of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD), for example of developing or worsening dementia, particularly if motor Parkinsonisian features or early signs of dementia are present.
  • TOMM40 genotype indicative of a higher probability of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD), for example of developing or worsening dementia, particularly if motor Parkinsonisian features or early signs of dementia are present.
  • the selected treatment regime may comprise treating the subject with, for example, one or more agents selected from agents considered to be useful in having a disease modifying effect on the PD and/or its non- motor complications, such as DLB or PDD, or in treating dementia, such as donezepil, Anti-TNF, B-MAO Inhibitors, Selegiline, Cyclosporine A and FK-506,
  • the TOMM40 genotype optionally in intron 6, optionally at rs10524523, is not heterozygous, optionally wherein there is no or less than 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3 or 2 base pair difference in the length of a poly-T stretch at rs 10524523 between the subject's alleles or wherein the subject's genotype at rs10524523 is not S VL, then the selected treatment regime may not require any additional components (for example over compounds that may be required more generally for treating PD/motor Parkinsonisian features).
  • the method may further comprise the step of performing tests to exclude other causes of dementia, such as carrying out a blood test for vitamin 12, as will be well known to those skilled in the art, or for other treatable metabolic causes. , Magnetic Resonance Imaging and metabolic imaging may also be performed.
  • a further aspect of the invention provides one or more agents selected from agents considered to be useful in having a disease modifying effect on the PD and/or its non- motor complications, such as DLB or PDD, or in treating dementia, such as donezepil, Anti-TNF, B-MAO Inhibitors, Selegiline, Cyclosporine A and FK-506, immunofilin ligands such as pentoxifylline and COX-2 Inhibitors, minocycline, Immunoglobulins, NMDA receptor antagonsists, PPAR agonists and modulators, iNOS Inhibitors, Copolymer-1 (Cop- ), GLP-1 receptor agonists, Acetylcholinesterase Inhibitors, other antibodies, fusion proteins, therapeutic RNA molecules and combination thereof, compounds referred to in lclat Aviles-Olmos et al, Brain 2012 or Tansey and Goldberg, Neurobiol.Dis 2010 or compounds mentioned at http://alzheimers.orq.uk site/scripts/
  • a further aspect of the invention provides the use of one or more agents selected from agents considered to be useful in having a disease modifying effect on the PD and/or its non-motor complications, such as DLB or PDD, or in treating dementia, such as donezepil, Anti-TNF, B- AO Inhibitors, Selegiline, Cyclosporine A and FK-506, immunofilin ligands such as pentoxifylline and COX-2 Inhibitors, minocycline, Immunoglobulins, NMDA receptor antagonsists, PPAR agonists and modulators, iNOS Inhibitors, Copolymer-1 (Cop-1), GLP-1 receptor agonists, Acetylcholinesterase Inhibitors, other antibodies, fusion proteins, therapeutic RNA molecules and combination thereof, compounds referred to in lclat Aviles-Olmos et al, Brain 2012 or Tansey and Goldberg, Neurobiol.Dis 2010 or compounds mentioned at http://alzheimers.org.uk/site/script
  • a further aspect of the invention provides a method for treating a subject with Parkinson's Disease or motor Parkinsonisian features or at risk thereof (other than through determination of TOMM40 genotype), the method comprising administering one or more agents selected from agents considered to be useful in having a disease modifying effect on the PD and/or its non-motor complications, such as DLB or PDD, or in treating dementia, such as donezepil, Anti-TNF, B-MAO Inhibitors, Selegiline, Cyclosporine A and FK-506, immunofilin ligands such as pentoxifylline and COX-2 Inhibitors, minocycline, Immunoglobulins, NMDA receptor antagonsists, PPAR agonists and modulators, iNOS Inhibitors, Copolymer-1 (Cop-1), GLP-1 receptor agonists, Acetylcholinesterase Inhibitors, other antibodies, fusion proteins, therapeutic RNA molecules and combination thereof, compounds referred to in lclat Aviles-Olmos et
  • the compound may alternatively (or in addition) be a modulator, for example inhibitor or activator of TOMM40, for example an antibody (including antibody fragment, as well known to those skilled in the art) or an RNAi molecule, directed at TOMM40 polypeptide or gene, as appropriate.
  • the compound may alternatively (or in addition) be a mitochondrial modulator or an anti-oxidant, such as Idebenone, a potent antioxidant and inhibitor of lipid peroxidation, interacting with the mitochondrial electron transport chain and facilitating mitochondrial electron flux in by-passing complex I (Haefeli et al., 2011 ) or EPI-743 (Sadun et al, Arch Neurol. 2012 Mar;69(3):331-8).
  • the compound may be a PPARy modulator, for example PPARy inhibitor or activator, for example piaglitazone, as discussed in, for example, Aviles-Olmos et al (2012), supra.
  • the subject may be administered an additional anti Parkinson's Disease or motor Parkinsonisian features treatment, for example selected from L Dopa with a peripheral dopa decarboxylase inhibitor, dopamine agonists, elective type B monoamine oxidase inhibitors, Amantadine, tolcapone and other agents.
  • an additional anti Parkinson's Disease or motor Parkinsonisian features treatment for example selected from L Dopa with a peripheral dopa decarboxylase inhibitor, dopamine agonists, elective type B monoamine oxidase inhibitors, Amantadine, tolcapone and other agents.
  • a further aspect of the invention provides a kit of parts useful for assessing a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features, comprising (1) an agent which is specifically capable of use in determining a subject's genotype at TOM 40, optionally intron 6, optionally rs 0524523.
  • the kit may contain PCR primers suitable for use in determining TOMM40 genotype, for example at position rs 0524523.
  • primers suitable for use in determining TOMM40 genotype for example at position rs 0524523.
  • examples of such primers include forward primer 5' -TGCTGACCTCAAGCTGTCCTC-3' and reverse primer 5'- GAGGCTGAGAAGGGAGGATT-3';
  • Such primers may be used for PCR amplification, typically followed by sequencing or separation of alleles by gel electrophoresis, for example capillary gel electrophoresis. Suitable sequence methods, including suitable internal controls and verification procedures, for example involving two independent PCR amplifications for each sample, are described in Example 1.
  • the kits may usefully further comprise a component for testing for a further PD/motor Parkinsonisian features related parameter, for example genes associated with familial PD (as noted above), or MAPT, gene or APOE.
  • kits usefully may contain controls and detection material, for example an internal control sequence, such as the sequence corresponding to a T8 poly-T sequence at position rs10524523, as described in Example 1 , "Addition of an Internal Standard" section.
  • an internal control sequence such as the sequence corresponding to a T8 poly-T sequence at position rs10524523, as described in Example 1 , "Addition of an Internal Standard" section.
  • a further aspect of the invention provides an agent which is specifically capable of use in determining a subject's genotype at TOM 40, optionally intron 6, optionally rs10524523, for aiding in determining likelihood of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD); or for categorising or determining prognosis, optionally a relatively high or relatively low likelihood of developing or worsening dementia, for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features; and/or in selecting a therapeutic strategy for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features.
  • DLB Lewy Bodies
  • PD Parkinson's Dementia
  • the subject may be a subject considered to have at least early motor Parkinsonisian features, as discussed above.
  • a further aspect of the invention provides the use of an agent which is specifically capable of use in determining a subject's genotype at TO M40, optionally intron 6, optionally rs10524523, in the manufacture of a medicament for aiding in determining likelihood of developing or worsening Parkinson's Disease, motor Parkinsonisian features, dementia with Lewy Bodies (DLB) or Parkinson's Dementia (PD); or for categorising or determining prognosis, optionally a relatively high or relatively low likelihood of developing or worsening dementia, for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features; and/or in selecting a therapeutic strategy for a subject with or otherwise at risk of Parkinson's Disease or motor Parkinsonisian features.
  • DLB Lewy Bodies
  • PD Parkinson's Dementia
  • the subject may be a subject considered to have at least early motor Parkinsonisian features, as discussed above.
  • the agent may typically be a nucleic acid which selectively hybridises to TOM 40 nucleic acid (typically genomic DNA), optionally in or flanking intron 6, optionally in or flanking rs 10524523 (for example within 100 bases either side of intron 6, or rs10524523).
  • the agent may be one or more of the PCR primers identified above or in Example 1.
  • nucleic acid has sufficient nucleotide sequence similarity with the said human nucleic acid that it can hybridise under moderately or highly stringent conditions.
  • stringency of nucleic acid hybridization depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and CG or AT content of the sequence.
  • any nucleic acid which is capable of selectively hybridising as said is useful in the practice of the invention.
  • TOMM40 sequences are well known, as indicated above and in Example 1.
  • Nucleic acids which can selectively hybridise to the said human nucleic acid include nucleic acids which have >95% sequence identity, preferably those with >98%, more preferably those with >99% sequence identity, over at least a portion of the nucleic acid with the said human nucleic acid.
  • human genes usually contain introns such that, for example, a mRNA or cDNA derived from a gene would not match perfectly along its entire length with the said human genomic DNA but would nevertheless be a nucleic acid capable of selectively hybridising to the said human DNA, other than intronic sequences.
  • Nucleic acids which span the intron-exon boundaries of the said TOMM40 gene may not be able to selectively hybridise to the said TOMM40 mRNA or cDNA but may be useful in the present invention for analysing intronic sequences.
  • Typical moderately or highly stringent hybridisation conditions which lead to selective hybridisation are known in the art, for example those described in Molecular Cloning, a laboratory manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, incorporated herein by reference.
  • the hybridisation is performed at 68 °C.
  • the nylon membrane, with the nucleic acid immobilised may be washed at 68°C in 1 x SSC or, for high stringency, 0.1 x SSC.
  • 20 x SSC may be prepared in the following way. Dissolve 175.3 g of NaCI and 88.2 g of Na + citrate in 800 ml of H 2 0. Adjust the pH to 7.0 with a few drops of a 10 N solution of NaOH. Adjust the volume to 1 litre with H 2 0. Dispense into aliquots. Sterilize by autoclaving.
  • An example of a typical hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe is an oligonucleotide of between 15 and 50 bases is:
  • the optimal temperature for hybridization is usually chosen to be 5 °C below the T, for the given chain length.
  • T is the irreversible melting temperature of the hybrid formed between the probe and its target sequence. Jacobs et al (1988) Nucl. Acids Res. 16, 4637 discusses the determination of TjS.
  • the recommended hybridization temperature for 17-mers in 3 M TMACI is 48-50 °C; for 19-mers, it is 55-57 °C; and for 20-mers, it is 58-66 °C.
  • nucleic acid which selectively hybridises is also included nucleic acids which will amplify DNA from the TOMM40 nucleic acid, typically genomic DNA, by any of the well known amplification systems such as those mentioned herein, in particular the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • nucleic acid which is useful in the methods of the invention may be RNA or DNA
  • DNA is preferred.
  • nucleic acid which is useful in the methods of the invention may be double-stranded or single-stranded, single-stranded nucleic acid is preferred under some circumstances such as in nucleic acid amplification reactions.
  • the nucleic acid which is useful in the methods of the invention may be any suitable size. However, for certain diagnostic, probing or amplifying purposes, it is preferred if the nucleic acid has fewer than 10 000, more preferably fewer than 000, more preferably still from 10 to 100, and in further preference from 15 to 30 base pairs (if the nucleic acid is double-stranded) or bases (if the nucleic acid is single stranded). As is described more fully below, single-stranded DNA primers, suitable for use in a polymerase chain reaction, are particularly preferred.
  • the nucleic acid for use in the methods of the invention is a nucleic acid capable of hybridising to the TOMM40 genomic DNA. Fragments of the said TOMM40 gene and are also preferred nucleic acids for use in the methods of the invention.
  • nucleic acid for use in the methods of the invention is an oligonucleotide primer which can be used to amplify a portion of the said TOMM40 nucleic acid, particularly TOMM40 genomic DNA.
  • Preferred nucleic acids for use in the invention are those that selectively hybridise to the TO M40 genomic DNA and do not hybridise to other related genomic DNA. Such selectively hybridising nucleic acids can be readily obtained, for example, by reference to whether or not they hybridise (or are predicted to hybridise, using well known calculations) to the said TOMM40 genomic DNA and not to other genomic sequences.
  • the nucleic acid capable of hybridising to the TOMM40 genomic DNA and which is used in the methods of the invention further comprises a detectable label, as discussed above.
  • a further aspect of the invention provides a method for selecting a test Parkinson's Disease or motor Parkinsonisian features or otherwise at risk thereof subject in whom to assess a therapeutic strategy or treatment regime for the treatment of Parkinson's Disease or motor Parkinsonisian features, which may include a therapeutic strategy or treatment regime intended for treating (for example preventing, delaying or reducing) dementia either in the early stages, i.e.
  • the method comprising the step of assessing the test subject's genotype for TOMM40, optionally in intron 6 (IVS6) of the TOMM40 gene, optionally at position rs10524523.
  • DLB year
  • PDD late stages
  • the method may comprise the step of determining whether the test subject's genotype for TOMM40, optionally in intron 6, optionally at rs10524523, is heterozygous, optionally determining whether there is a difference in the length of a poly- T stretch at rs10524523 between the subject's alleles, optionally whether the subject's genotype at rs10524523 is S/VL or whether the difference in the length of the poly-T stretch is more than or equal to 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 base pairs, optionally more than or equal to 12, 13, 14, 15, 16, 17 or 18 base pairs, for example more than or equal to 15 base pairs.
  • the subject typically is a human subject.
  • the subject may be a subject considered to have at least early motor Parkinsonisian features, as discussed above
  • the test subject may be selected if the TOMM40 genotype, optionally in intron 6, optionally at rs10524523, is heterozygous, optionally wherein there is a difference in the length of a poly-T stretch at rs10524523 between the subject's alleles, optionally wherein the subject's genotype at rs10524523 is S/VL or wherein the difference in the length of the poly-T stretch is more than or equal to 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 base pairs, optionally more than or equal to 12, 13, 14, 15, 16, 17 or 18 base pairs, for example more than or equal to 15 base pairs.
  • the therapeutic strategy or treatment regime may be assessed both in subjects selected as indicated above; and in subjects selected as not meeting that criterion; and the results obtained in the two subject groups compared.
  • a further aspect of the invention provides a method for assessing a therapeutic strategy or treatment regime for the treatment of Parkinson's Disease or motor Parkinsonisian features (which may include a therapeutic strategy or treatment regime intended for treating (for example preventing, delaying or reducing) PDD or DLB), or at risk thereof (other than through determination of TOMM40 genotype), the method comprising the step of evaluating the results of the therapeutic strategy or treatment regime from a subject or subjects who have been selected according to the method of the preceding aspect of the invention.
  • a further aspect of the invention provides an in vitro method for assessing the suitability of a test compound for the treatment of a subject with or at risk of Parkinson's Disease, motor Parkinsonisian features, PDD or DLB, the method comprising the step of determining the effect of the test compound on a cell for which it has been determined that the TOMM40 genotype, optionally in intron 6, optionally at rs10524523, is heterozygous, optionally wherein there is a difference in the length of a poly-T stretch at rs10524523 between the cell's alleles, optionally wherein the cell's genotype at rs10524523 is S/VL or wherein the difference in the length of the poly-T stretch is more than or equal to 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs, optionally more than or equal to 12, 13, 14, 15, 16, 17 or 18 or more base pairs.
  • An assay may also be in an animal model of Parkinson's Disease, for example a transgenic, neurotoxin or virus-based animal model of PD, as known to those skilled in the art.
  • a further aspect of the invention provides a method for assessing the suitability of a test compound for the treatment of a subject with or at risk of Parkinson's Disease, motor Parkinsonisian features, PDD or DLB, the method comprising the step of determining the effect of the test compound on a cell in a non-human animal for which it has been determined that the TOMM40 genotype, optionally in intron 6, optionally at rs10524523, is heterozygous, optionally wherein there is a difference in the length of a poly-T stretch at rs10524523 between the animal's alleles, optionally wherein the animal's genotype at rs10524523 is S VL or wherein the difference in the length of the poly-T stretch is more than or equal to 10, 11 , 12, 13, 14, 15, 16, 17, 18, 9 or 20 base pairs, optionally more than or equal to 12, 13, 14, 15, 16, 17 or 18 base pairs, for example more than or equal to 15 base pairs.
  • a further aspect of the invention provides a screening method for aiding in identifying a compound likely to be useful in treating a subject with Parkinson's Disease or motor Parkinsonisian features or at risk thereof, the method comprising the step of determining the effect of a test compound on TO M40 nucleic acid, protein or activity level; and selecting a compound that modulates, for example reduces or increases, said level.
  • This screening method may be performed on a test compound together with (either before or after) the method of the preceding aspect of the invention.
  • the effect of the test compound may be determined in vitro.
  • test compound may be determined in vivo in a non-human test animal.
  • Fig 1. Distribution of the TOMM40 rs10524523 allele length, and their clustering in three groups, following Lutz et al. 2010 16 .
  • A Distribution of TOMM40 rs10524523 absolute allele length differences over the three clinical phenotypes. The dashed line represents a difference of 15, the minimum difference observed in the S VL genotype, and not observed in any other genotype.
  • Figure 3 analysis of how Positive Predictive Value (PPV) and Negative Predictive Value (NPV) change across the allele lengths difference (ALD) spectrum.
  • PPV Positive Predictive Value
  • NPV Negative Predictive Value
  • ALD allele lengths difference
  • H1/H2 MAPT haplotypes based on four SNPs (rs242557, rs3785883, rs247 738 and rs9468), and differentiated APOE ⁇ 2/ ⁇ 3/ ⁇ 4 genotypes based on two SNPs (rs429358 and rs7412).
  • the IVS6 poly-T repeats are in Hardy-Weinberg equilibrium (p- value of 1 for the raw data after 10,000,000 replicates, and p-value of 0.68 after 10000 replicates).
  • the results from a Cox proportional hazards model showed that the S/VL TOMM40 rs10524523 genotype was strongly associated with dementia, with an odds ratio (OR) of 3.83 (p value 0.002).
  • OR odds ratio
  • the positive and negative predictive values of a TOMM40 ISV6 poly-T S/VL genotype are 25% and 95%, respectively, suggesting that a patient in the very early stages of PD found to harbor this genotype would carry a risk of 25% of developing DLB, whereas a patient found not to carry this genotype would have a 95% chance of not developing early dementia.
  • rs10524523 may have clinical utility in predicting risk estimates of early dementia in the very early stages of Parkinsonism.
  • TOM 40 is both strongly associated with and predictive of DLB in a population sample of PD patients.
  • Our findings represent an important novel insight with potential implications both in the clinical setting suggesting that the TOMM40 IVS6 polyT may be a powerful predictive biomarker of early dementia in PD and, by pointing towards a possible pathogenic role of perturbations of mitochondrial function and dynamics, for drug discovery of novel disease modifying therapeutics.
  • the complete methods are provided in the Supplementary Methods and Data, which include clinical, neuropathological and genotypic data, data quality controls, and detailed statistical methods.
  • complete genotyping data were obtained in 218 cases.
  • the clinical and neuropathological records were reviewed and 120 cases were selected based on neuropathological diagnosis of PD and reliable clinical information of presence or absence of dementia, with the characteristic features of visual hallucinations and fluctuating confusion.
  • a Cox proportional hazard model was used to analyze the effects of genotype, adjusting for age of onset of motor disease, vascular risk score and stratifying the model for gender.
  • Table 1 A: Cox proportional hazards model stratified by gender, including TOMM40 rs10524523 (baseline risk S/S), APOE (baseline risk ⁇ 3/ ⁇ 3), MAPT (baseline risk H2/H2), age of onset and Vascular Risk Score as predictors.
  • Model A model stratified by Sex
  • VL/VL 1.45 0.54 3.87 0.461 ⁇ 2/ ⁇ 3 0.44 0.18 1.07 0.071 ⁇ 2/ ⁇ 4 2.20 0.35 13.91 0.403 ⁇ 3/ ⁇ 4 0.94 0.40 2.17 0.876 ⁇ 4/ ⁇ 4 2.51 0.64 9.82 0.186
  • Model B model stratified by Sex
  • Table 2 Confusion matrix for TOMM40 allele length difference, with the resulting sensitivity, specificity, positive predictive value and negative predictive value for a 15 bases allele length difference threshold (see supplementary material, picture 1).
  • Table 3 Demographic, clinical and neuropathological data of Parkinson's disease patients classified according to presence/absence of dementia and dementia type.
  • DLB dementia with Lewy bodies;
  • PDD Parkinson's disease with Dementia;
  • PDnD 10 Parkinson's disease with no dementia.
  • aSN Alpha-synuclein
  • AC Anterior Cingulate
  • PC Parietal Cortex
  • RBD REM-sleep Behaviour Disorder
  • SFC Superior Frontal Cortex
  • TC Temporal Cortex.
  • VL VL 0.91 2.33 0.39 0.70
  • VL/VL 0.91 2.33 0.39 0.70
  • Cox model stratified by sex including TOMM40 rs10524523 (baseline risk S/S), APOE (baseline risk ⁇ 3/ ⁇ 3), MAPT (baseline risk H2/H2), Age of Onset and Vascular Risk Score as predictors. - PDnD and PDD only, excluding DLB
  • Table 8 Cox model for TOMM40 '523 allele (baseline risk S/S), Age of Onset and0 Vascular Risk Score as predictors. Model stratified by sex.
  • VLA L 1.44 0.54 3.81 0.464
  • Table 9 Cox model for APOE (baseline risk ⁇ 3/ ⁇ 3), Age of Onset and Vascular Risk5 Score as predictors. Model stratified by sex.
  • Table 10 Cox model for MAPT (baseline risk H2/H2), age of onset and Vascular Risk Score as predictors. Model stratified by sex.
  • Clinical summaries were extrapolated for each patient by reviewing medical records, which included neurological, psychiatric, and geriatric examinations, GP printouts and notes, formal cognitive testing and neuroimaging results.
  • Clinical records were assessed by four neurologists (SM, PP, C.R., L.M.) and 20 randomly selected cases were subsequently reviewed by two neurologists with expertise in movement disorders (SM and PP), blinded to one another's evaluation, to confirm diagnosis.
  • Presence of dementia was ascertained based on DSM IV criteria and was defined as a decline from a preceding normal status in more than one cognitive domain causing a significant impairment in activities of daily living 3 .
  • Cognitive testing was also used when available: scores equal to or below 24/30 on the standard Mini Mental State Examination or equal to or below 7/10 on the Abbreviated Mental Test (AMT) were considered diagnostic of dementia 4,5 .
  • AMT Abbreviated Mental Test
  • Dysautonomia was scored by adding reported dysautonomic symptoms among the following: constipation; bladder impairment including urinary urgency/frequency, nocturia, and retention; orthostatic hypotension; erectile dysfunction; altered perspiration; hypersalivation; dysphagia.
  • vascular risk As an indirect measure of a possible contribution of vascular impairment to the clinical picture, we scored vascular risk by adding one point for each risk factor among the following, when they were reported in medical records: systemic hypertension, ischaemic heart disease, myocardial infarction, arrythmia (mainly atrial fibrillation), unequivocal family history of vascular disease, hyperlipidaemia, smoker status, neuroimaging reports (CT or MRI) of small vessel disease, clinical or neuroimaging reports of cerebrovascular accidents (stroke or transient ischaemic attacks).
  • CT or MRI neuroimaging reports of small vessel disease
  • Neuropathological diagnosis of Parkinson's disease was based on presence of Lewy bodies associated with neuronal loss in the substantia nigra, in accordance with accepted criteria 11 .
  • Diagnosis of dementia with Lewy bodies was based on the same neuropathological findings, in association with a clinical history of dementia occurring within one year of onset of parkinsonism 2 .
  • Topographical distribution of LB and LD was defined according to recently proposed guidelines. Briefly, presence or absence of alpha-synuclein-immunoreactive LBs or LDs was assessed in nine brain regions, with stages 1 to 6 identifying progression of pathology from lower brainstem to neocortical regions 12,13 .
  • stage A corresponds to tau-positive neuropil threads limited to the trans-entorhinal and/or entorhinal regions
  • stage B to involvement of the occipito-temporal and/or middle temporal gyri
  • stage C to spread of neuropil threads to the occipital peristriate and/or striate areas, with a progression of severity of pathology from A to C.
  • Genotyping for APOE and the TOMM40 rs10524523 poly T repeat was carried out by Polymorphic DNA Technologies using Sanger dideoxy DNA sequencing 17 Polymorphic genotyped two SNPs (rs429358, rs7412) used to identify APOE ⁇ 2/ ⁇ 3/ ⁇ 4 haplotypes, and genotyped the APOE poly T repeat. Genotyping for MAPT and again for APOE was carried out by Newgene, using Sequenom MALDI-TOF technology 13 .
  • Newgene genotyped 6 MAPT SNPs (rs242557, rs1800547, rs3785883, rs 98 997, rs2471738 and rs9468) which allowed to determine MAPT H1/H2 haplotypes, and the same APOE SNPs (rs429358, rs7412) as Polymorphic.
  • TOMM40 IVS6 rs10524523 poly T repeats were classified as 'Short' (poly T repeat shorter than 19 Ts), 'Long' (poly T repeat of 19 to 29 Ts) and 'Very Long' (poly T repeat longer than 30 Ts) 19 . For each individual we also calculated the allele length difference and the mean pair length. Assay description of the high-throughput sequencing and analytical validity data for TOMM40 rs10524523 (Polymorphic DNA Technologies, Inc.):
  • Polymorphic DNA Technologies, Inc. (Alameda CA, polymorphicdna.com) developed two independent assays that use PCR amplification of the region surrounding located at chrl 9:45,403,049-45,403,083 (hg19) in the intron between the 5 th and 6 th coding exons of the gene TOMM40 (mitochondrial import receptor subunit Tom40 homolog). In both cases the PCR amplification step was followed by Sanger sequencing of the PCR templates. The two independent assays are labeled "Assay 1" and "Assay 2".
  • the oligonucleotide primers used in Assay 2 are different from those used in Assay 1 , so that any unknown variation at a primer site for one assay will not interfere with the amplification in the other assay.
  • Sanger sequencing single-base resolution can be achieved using the automation and reliability of the ABI 3730x1 sequencing platform.
  • This platform also provides reference sequence information that confirms the identity of the amplified region and also gives contextual information that can be used to estimate the exact sizes of the homopolymer.
  • PCR Primers In order to achieve a high level of specific amplification a two-step, nested PCR strategy was used to amplify the region flanking '523. Human genomic DNA samples are first PCR-amplified with a pair of "outer” oligonucleotide primers, and those amplification products are then re-amplified with a second pair of "inner” primers.
  • the primers used in Assay 1 for these amplifications are as follows:
  • the resulting final PCR product maps to the human reference sequence in the range chrl 9:45,402,906-45,403, 151.
  • the reference sequence of the amplicon produced in Assay 2 is shown below:
  • the 3'-ends were chosen to be in locations in which there are no A-residues.
  • PCR Procedures The first "outer" PCR reaction was performed in a 384-well plate by combining in each well approximately 10 ng of genomic DNA with 1 unit of Klen Taq (Ab Peptides, Inc.), 0.5 pL of 10 X PCR Buffer, 1.0 pL of 5.0 M betaine, 0.175 ⁇ _ DMSO, 0.8 pL of a mixture of dNTPs (2.5 mM each), 0.5 pl_ of a mixture of the two outer oligonucleotide primers (2 ⁇ each), plus water to bring the entire reaction volume per well to a total of 5.0 ⁇ _. Plates were placed in a thermocycler and subjected to 40 cycles of the following conditions: 94° C for 20 s., 55° C for 25 s., and 72° C for 60 s.
  • the second "inner" PCR reaction was performed in a different 384-well plate by combining in each well 0.8 ⁇ _ of the product from the first PCR reaction with 0.25 units of Klen Taq, 0.5 pL of 10 X PCR Buffer, 1.0 pL of 5.0 M betaine, 0.175 ⁇ _ DMSO, 0.2 ⁇ _ of a mixture of dNTPs (2.5 mM each), 1.0 ⁇ _ of a mixture of the two inner oligonucleotide primers (2 ⁇ each), plus water to bring the entire reaction volume per well to a total of 5.0 pL.
  • This cloned T8 sequence containing all oligonucleotide priming sites used in both Assay 1 and Assay 2 was amplified with the same primers and under the same conditions in order to create an appropriate internal standard for each assay.
  • an aliquot of the appropriate internal standard amplification product was added in the ratio 8:1 (8 parts amplicon from the genomic sample to 1 part amplicon from the plasmid clone.)
  • Method Validation The method has been validated in three ways. Firstly, the method was applied to cloned DNA with known poly-T sizes and this method confirms the sizes of those poly-T repeats. Secondly, the method was run on genomic DNA samples for which the poly-T genotypes had been independently measured by the sequencing of clones from long-range PCR. In this case, the present method gives results that are the same within the statistical uncertainty of the clones picked. Thirdly, the allele distribution as defined by this method was found to be comparable to that found by cloning methods, again with the caveat that this was identified to result from those cloning methodologies. Statistical methods
  • the TOMM40 IVS6 rs10524523 fragment lengths was classified in three groups, short (S), long (L) and very long(VL) 19 .
  • the distribution of fragment length strongly suggests that three ciades are present, and the subdivision of fragment lengths into three groups reflects this observation.
  • Our first analysis used a Cox Proportional Hazard model to model the effects of TOMM40 rs10524523, and the effects of APOE, MAPT, age of onset of motor symptoms and vascular risk score. While the Cox model only takes presence or absence of dementia as a variable, it also automatically adjusts for latency to dementia, making the subdivision between DLB and PDD redundant for this kind of model.
  • the strong association between the most strongly heterozygous genotype and dementia raised the issue of whether the binning of rs10524523 fragment length could actually dilute some of the effects of rs10524523 on dementia status.
  • the first is an absolute value representing the difference between the two allele lengths (ALD) and is directly related to the level of heterozygosity between the two alleles. Closely or perfectly matched alleles would yield a low score, while a high score would reflect highly divergent allele lengths.
  • the second measure that we used was the mean fragment length. This measure is directly related to both homozygosity and to overall length concordance.
  • Sensitivity, specificity, positive predictive value and negative predictive value were calculated from the confusion matrix in Table 2, using the R package 'Caret' 20 and by double checking the results by doing the calculations by hand 21,22 .
  • Table 11 list of 17 individuals showing either severe A(3deposition in the frontal cortex and/or Braak Tau stage V VI. Highlighted are 4 cases with possible concurrent AD pathology
  • Table 12 Cox model stratified by sex, including TO M40 rs10524523 (baseline risk S/S), APOE (baseline risk ⁇ 3/ ⁇ 3), MAPT (baseline risk H2/H2), age of onset and Vascular Risk Score as predictors.
  • A Cox model stratified by sex, including TO M40 rs10524523 (baseline risk S/S), APOE (baseline risk ⁇ 3/ ⁇ 3), MAPT (baseline risk H2/H2), age of onset and Vascular Risk Score as predictors.
  • B Cox model including TOMM40 '523 allele length difference, TOMM40 rs10524523 mean pair length, APOE (baseline risk ⁇ 3/ ⁇ 3), MAPT 5 (baseline risk H2/H2), age of onset and Vascular Risk Score as predictors
  • Model A model stratified by Sex
  • Model B model stratified by Sex
  • Alzheimer's disease 16 Mirra, S.S. et al. The Consortium to Establish a Registry for Alzheimer's disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology 1991; 41: 479-486.
  • Example 2 Clinical trial of candidate Parkinson's Disease treatment
  • the candidate compound is assessed in Parkinson's Disease patients (or patients considered to be at risk of Parkinson's Disease) selected on the basis of a relatively high likelihood of rapid development of dementia based on their TO 40 genotype, possibly combined with other factors such as sex (male). Because the selected patients have a relatively high likelihood of rapid development of dementia, the effect (or lack of effect) of the candidate compound should be apparent much earlier in such patients than in unselected PD patients, where the overall likelihood of rapid development of dementia would be much lower. This means that the trial can potentially be conducted more quickly and with fewer patients.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour aider à déterminer la susceptibilité de développer ou exacerber la maladie de Parkinson, des caractéristiques Parkinsoniennes motrices, une démence avec des corps de Lewy (DLB) ou la démence de Parkinson (PD) ; ou pour catégoriser ou déterminer le pronostic, éventuellement une susceptibilité relativement élevée ou relativement faible de développer ou exacerber la démence, pour un sujet atteint ou présentant autrement un risque de la maladie de Parkinson ou des caractéristiques Parkinsoniennes motrices ; et/ou pour sélectionner une stratégie thérapeutique pour un sujet atteint ou présentant autrement un risque de maladie de Parkinson ou de caractéristiques Parkinsoniennes motrices, le procédé comprenant l'étape d'évaluation du génotype du sujet pour TOMM40, facultativement dans l'intron 6 (IVS6)du gène TOMM40, facultativement à la position rs10524523. Le procédé peut comprendre l'étape de détermination de savoir si le génotype du sujet pour TOMM40, éventuellement dans l'intron 6, éventuellement au niveau de rs10524523, est hétérozygote, éventuellement de détermination de savoir s'il existe une différence dans la longueur de l'étirement poly-T au niveau de rs10524523 entre les allèles du sujet, éventuellement si le génotype du sujet de rs10524523 est SA/L ou si la différence dans la longueur de l'étirement poly-T est supérieure ou égale à 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ou 20 paires de bases, éventuellement supérieur ou égal à 12, 13, 14, 15, 16, 17 ou 18 paires de bases. Si le génotype TOMM40, éventuellement dans l'intron 6, éventuellement au niveau de rs10524523 est hétérozygote, éventuellement s'il y a une différence dans la longueur d'un étirement poly-T au niveau de rs10524523 entre les allèles du sujet, éventuellement si le génotype du sujet au niveau de rs10524523 est SA/L ou si la différence dans la longueur de l'étirement poly-T est supérieure ou égale à 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ou 20 paires de bases, éventuellement supérieure ou égale à 12, 13, 14, 15, 16, 17 ou 18 paires de bases, alors le sujet est considéré comme présentant un risque supérieur de développer ou exacerber la maladie de Parkinson, des caractéristiques Parkinsoniennes motrices, une démence avec des corps de Lewy (DLB) ou la démence de Parkinson (PD) ; ou présentant un risque supérieur de progression de la maladie, éventuellement un risque supérieur de développer la démence de la maladie de Parkinson (PDD), éventuellement une démence avec des corps de Lewy (DLB), éventuellement de développer DLB en une période de un à deux ans de l'apparition de symptômes moteurs de la maladie de Parkinson ou de caractéristiques Parkinsoniennes motrices.
PCT/GB2013/051463 2012-05-31 2013-05-31 Tomm40 en tant que marqueur pour la maladie de parkinson WO2013179061A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1209689.7 2012-05-31
GB201209689A GB201209689D0 (en) 2012-05-31 2012-05-31 Methods

Publications (1)

Publication Number Publication Date
WO2013179061A1 true WO2013179061A1 (fr) 2013-12-05

Family

ID=46582124

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2013/051463 WO2013179061A1 (fr) 2012-05-31 2013-05-31 Tomm40 en tant que marqueur pour la maladie de parkinson

Country Status (2)

Country Link
GB (1) GB201209689D0 (fr)
WO (1) WO2013179061A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230085582A (ko) * 2021-12-07 2023-06-14 주식회사 엔젠바이오 루이소체 치매 진단을 위한 단일염기다형성 마커
KR20230085588A (ko) * 2021-12-07 2023-06-14 주식회사 엔젠바이오 파킨슨병 치매 진단을 위한 단일염기다형성 마커

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092576A1 (fr) * 2000-05-26 2001-12-06 Duke University Methodes de depistage de la maladie de parkinson
WO2009086202A2 (fr) * 2007-12-19 2009-07-09 Afraxis, Inc. Procédés de traitement d'affections neuropsychiatriques
WO2010019550A2 (fr) * 2008-08-12 2010-02-18 Shiraz Pharmaceuticals, Inc. Procédé d'identification de facteurs de risque de maladie
US20110166185A1 (en) * 2008-08-12 2011-07-07 Zinfandel Pharmaceuticals, Inc. Disease risk factors and methods of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092576A1 (fr) * 2000-05-26 2001-12-06 Duke University Methodes de depistage de la maladie de parkinson
WO2009086202A2 (fr) * 2007-12-19 2009-07-09 Afraxis, Inc. Procédés de traitement d'affections neuropsychiatriques
WO2010019550A2 (fr) * 2008-08-12 2010-02-18 Shiraz Pharmaceuticals, Inc. Procédé d'identification de facteurs de risque de maladie
US20110166185A1 (en) * 2008-08-12 2011-07-07 Zinfandel Pharmaceuticals, Inc. Disease risk factors and methods of use

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDREAS BENDER ET AL: "TOM40 Mediates Mitochondrial Dysfunction Induced by [alpha]-Synuclein Accumulation in Parkinson's Disease", PLOS ONE, vol. 8, no. 4, 23 April 2013 (2013-04-23), pages e62277, XP055079722, DOI: 10.1371/journal.pone.0062277 *
BEATA PEPLONSKA ET AL: "TOMM40 and APOE common genetic variants are not Parkinson's disease risk factors", NEUROBIOLOGY OF AGING, vol. 34, no. 8, 1 August 2013 (2013-08-01), pages 2078.e1 - 2078.e2, XP055079723, ISSN: 0197-4580, DOI: 10.1016/j.neurobiolaging.2013.02.018 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230085582A (ko) * 2021-12-07 2023-06-14 주식회사 엔젠바이오 루이소체 치매 진단을 위한 단일염기다형성 마커
KR20230085588A (ko) * 2021-12-07 2023-06-14 주식회사 엔젠바이오 파킨슨병 치매 진단을 위한 단일염기다형성 마커
KR102737914B1 (ko) 2021-12-07 2024-12-04 주식회사 엔젠바이오 파킨슨병 치매 진단을 위한 단일염기다형성 마커
KR102737915B1 (ko) 2021-12-07 2024-12-04 주식회사 엔젠바이오 루이소체 치매 진단을 위한 단일염기다형성 마커

Also Published As

Publication number Publication date
GB201209689D0 (en) 2012-07-18

Similar Documents

Publication Publication Date Title
DeJesus-Hernandez et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS
JP6338530B2 (ja) アルツハイマー病を治療、診断、および監視するための方法
Macé et al. ABCA2 is a strong genetic risk factor for early-onset Alzheimer's disease
Kwok et al. Tau haplotypes regulate transcription and are associated with Parkinson's disease
JP5917460B2 (ja) アルツハイマー病の診断および治療
Xi et al. Jump from pre-mutation to pathologic expansion in C9orf72
US20200270692A1 (en) Predicting age-related macular degeneration with single nucleotide polymorphisms within or near the genes for complement component c2, factor b, plekha1, htra1, prelp, or loc387715
US20100028356A1 (en) Method of diagnosing a neurodegenerative disease
US20090075827A1 (en) Diagnostic methods and agents
US20090041862A1 (en) Detecting disease association with aberrant glycogen synthase kinase 3beta expression
Shi et al. Mutation screening and association study of the neprilysin gene in sporadic Alzheimer's disease in Chinese persons
WO2009112882A1 (fr) Biomarqueurs de snp pour la maladie d'alzheimer
US20240026454A1 (en) Single nucleotide polymorphism marker for predicting risk of alzheimers disease and use thereof
US20080118918A1 (en) Compositions and Methods For Determining and Predicting Treatment Responses For Depression and Anxiety
WO2013179061A1 (fr) Tomm40 en tant que marqueur pour la maladie de parkinson
Venturelli et al. The NOS3 G894T (Glu298Asp) polymorphism is a risk factor for frontotemporal lobar degeneration
US20130324431A1 (en) Olfactory receptor copy number association with age at onset of alzheimer's disease
EP1863937B1 (fr) Gene humain de susceptibilite a l'autisme codant une proteine transmembrane et ses utilisations
US20070298027A1 (en) Novel Target for Antiderpressant Therapy
Todd et al. BACE1 polymorphisms do not influence platelet membrane β-secretase activity or genetic susceptibility for Alzheimer’s disease in the northern Irish population
Burdon et al. Investigation of eight candidate genes on chromosome 1p36 for autosomal dominant total congenital cataract
JP2022150707A (ja) アミロイドβ蓄積リスクの有無の指標とする方法、一塩基多型（ＳＮＰ）の存在又は非存在を検出する方法、組成物、及びキット
Martinez-Lage Neurodegenerative Disorders
Al-Saud The genetics of obesity in Saudi Arabian population

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13735360

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13735360

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载