WO2013131981A1 - Marqueurs de prédiction utiles dans le diagnostic et le traitement du syndrome de l'x fragile (fxs) - Google Patents

Marqueurs de prédiction utiles dans le diagnostic et le traitement du syndrome de l'x fragile (fxs) Download PDF

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WO2013131981A1
WO2013131981A1 PCT/EP2013/054542 EP2013054542W WO2013131981A1 WO 2013131981 A1 WO2013131981 A1 WO 2013131981A1 EP 2013054542 W EP2013054542 W EP 2013054542W WO 2013131981 A1 WO2013131981 A1 WO 2013131981A1
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chromosome
genomic locus
fxs
hydroxy
hydroxymethylation
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PCT/EP2013/054542
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English (en)
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Remi TERRANOVA
Baltazar Gomez-Mancilla
Jonathan MOGGS
Olivier Grenet
Florian HAHNE
Sarah BRASA
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Novartis Ag
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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
    • C12Q2600/154—Methylation markers

Definitions

  • FXS Fragile X Syndrome
  • the present invention provides methods and compositions directed to identification of genetic and epigenetic markers associated with Fragile X syndrome (FXS) disorders.
  • FXS Fragile X syndrome
  • Fragile X syndrome is the most common cause of inherited mental retardation with a worldwide prevalence of 1/4000 in males and 1/8000 in females. The incidence of FXS is 10-20 times higher than other X-linked mental retardations.
  • FXS is a monogenetic disease and mainly caused by a CGG-repeat expansion that triggers hypermethylation and silencing of the fragile X mental retardation 1 (FMRl) gene.
  • FMRl fragile X mental retardation 1
  • FMRP FMRl protein
  • mGluR5 antagonists have the potential to reduce the mGluR5 signaling and normalize the deficits caused by the lack of fragile X mental retardation protein.
  • SSRIs selective serotonin reuptake inhibitors
  • alpha-adreno-receptor agonists e.g., clonidine
  • mood stabilizers e.g. carbamazepine
  • antipsychotic medication e.g., risperidone, olazapine
  • FMRl epigenetic silencing in FXS are still elusive and their characterization may enhance the development of novel clinical biomarkers of disease and/or drug response as well as support the identification of novel therapeutic targets. This would contribute as well to the identification of epigenetic biomarkers for selecting a sub-population of FXS patients that might respond to particular FXS therapies.
  • the present invention is based on the finding of novel regions of epigenetic modifications within the FMRl genomic locus. Specifically, two methylation markers, DNA methylation and DNA
  • the invention includes a method for the diagnosis of Fragile X syndrome (FXS) or a predisposition for FXS including detecting at an epigenetic biomarker selected from DNA methylation or hydroxymethylation at a predetermined region on chromosome X of an FMR1 genomic locus, and comparing the amount of methylation or hydroxymethylation to a control, whereby FXS or the predisposition for FXS can be diagnosed.
  • FXS Fragile X syndrome
  • a predisposition for FXS including detecting at an epigenetic biomarker selected from DNA methylation or hydroxymethylation at a predetermined region on chromosome X of an FMR1 genomic locus, and comparing the amount of methylation or hydroxymethylation to a control, whereby FXS or the predisposition for FXS can be diagnosed.
  • the predetermined chromosome X region for detecting DNA methylation can be between position 146993800 to 147048300 (herein coordinates based on Human reference genome GRCh37/hgl9), e.g., any predetermined chromosome X region as shown in Table 1, 2 or 3, or portion thereof.
  • one or more of the DNA methylation regions shown in Table 1 can be assayed.
  • the predetermined region for detecting DNA methylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the predetermined chromosome X region for detecting DNA hydroxymethylation can be between 146982000 to 147027400 (herein coordinates based on Human reference genome GRCh37/hgl9), e.g., any predetermined chromosome X region as shown in Table 1, 2 or 3, or portion thereof.
  • one or more of the hydroxymethylation regions shown in Table 1 can be assayed.
  • the predetermined region for hydroxymethylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the invention includes a method for the diagnosis of Fragile X syndrome (FXS) or a predisposition for FXS including detecting at least two epigenetic biomarkers selected from DNA methylation and hydroxymethylation at a predetermined region on chromosome X of an FMR1 genomic locus, comparing the amount of hydroxymethylation to a control, whereby FXS or the predisposition for FXS can be diagnosed.
  • the predetermined chromosome X region for detecting DNA methylation can be between position 146993800 to 147048300 and for hydroxymethylation is between 146982000 to 147027400 (herein coordinates based on Human reference genome GRCh37/hgl9).
  • a diagnostic or prognostic determination can be made based on the amount of DNA methylation compared to a control and amount of hydroxymethylation compared to a control.
  • the predetermined region for detecting DNA methylation includes one or more of the regions shown in Table 1 , 2 or 3 (regions listed under "coordinates") and the predetermined region for detecting hydroxymethylation includes one or more of the regions shown in Table 1, 2 or 3 (regions listed under "coordinates").
  • the predetermined region for detecting DNA methylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the predetermined region for detecting hydroxymethylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the predetermined region for detecting DNA methylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the predetermined region for detecting hydroxymethylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the invention includes a method for the diagnosis of Fragile X syndrome (FXS) or a predisposition for FXS comprising detecting DNA methylation in a sample, e.g., Peripheral Blood Mononuclear Cells (PBMC), comprising one or more of the following predetermined regions:
  • FXS Fragile X syndrome
  • PBMC Peripheral Blood Mononuclear Cells
  • the invention includes a method for the diagnosis of Fragile X syndrome (FXS) or a predisposition for FXS comprising detecting hydroxymethylation in a sample, e.g., PBMC comprising one or more of the following predetermined regions:
  • the invention includes a method for the diagnosis of Fragile X syndrome (FXS) or a predisposition for FXS comprising detecting hydroxymethylation in a sample, e.g., PBMC comprising one or more of the following predetermined regions:
  • the invention includes a method for determining responsiveness of an individual with Fragile X Syndrome (FXS) to treatment with an mGluR antagonist, the method comprising:
  • the predetermined region for detecting DNA methylation comprises one or more of the following regions:
  • the predetermined region for detecting 5- hydroxymethylcytosine comprises one or more of the following regions:
  • the invention includes a method for determining responsiveness of an individual with Fragile X Syndrome (FXS) to treatment with a test molecule such as an mGluR antagonist, the method comprising:
  • the predetermined region comprises one or more of the following regions:
  • the individual is selected for treatment with an mGluR antagonist such as (-)-(3aR, 4S, 7aR)-4-Hydroxy-4- m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof.
  • an mGluR antagonist such as (-)-(3aR, 4S, 7aR)-4-Hydroxy-4- m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof.
  • the invention includes a method for determining responsiveness of an individual with Fragile X Syndrome (FXS) to treatment with an mGlur5 antagonist, the method comprising:
  • a sample e.g., PBMC, from an individual having Fragile X Syndrome; and detecting hydroxymethylation at a predetermined region in the sample, wherein the
  • predetermined region comprises one or more of the following regions:
  • the mGlur5 antagonist can be any known mGluR5 antagonist such as (-)-(3aR, 4S, 7aR)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-l- carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof.
  • the predetermined region comprises one or more of the following regions:
  • the invention includes a method of selecting an individual with Fragile X
  • FXS Factorized Hass syndrome
  • the method comprising detecting hydroxymethylation at a predetermined region in the sample, wherein the predetermined region comprises one or more of the following regions:
  • the individual is selected for treatment on the basis of the subject having an amount of hydroxymethylation indicative that the individual will respond to treatment with (-)-(3aR, 4S, 7aR)-4- Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof.
  • the method can further include administering (-)-(3aR, 4S, 7aR)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, or a
  • the method can further include administering a compound other than (-)-(3aR, 4S, 7aR)-4-Hydroxy-4-m-tolylethynyl- octahydro-indole-l-carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof to an individual on the basis of that individual on the basis of the individual not having an amount of hydroxymethylation compared to a control indicative that the individual will respond to treatment with (-)-(3aR, 4S, 7aR)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester.
  • the invention includes a method of selectively treating a subject having FXS, comprising selectively administering a therapeutically effective amount of ( ⁇ -Pyrrolidine- 1,2- dicarboxylic acid 2-amide 1 -( ⁇ 4-methyl-5-[2-(2,2,2-trifluoro- 1 , 1 -dimethyl-ethyl)-pyridin-4-yl]-thiazol- 2-yl ⁇ -amide), or a pharmaceutically acceptable salt thereof, to the subject on the basis of the amount of hydroxymethylation compared to a control at a predetermined region in the sample, wherein the predetermined region comprises one or more of the following regions:
  • the invention includes a method of screening for an agent that modulates an epigenetic biomarker including contacting an agent with a mammalian cell; and detecting at least two biomarkers selected from DNA methylation and hydroxymethylation at a predetermined region as described in Table 1 or 3 within the FMRl genomic locus, wherein a change in biomarker status relative to a control is indicative that the agent is an epigenetic biomarker modulating agent.
  • the invention includes an mGluR5 antagonist, e.g., (-)-(3aR, 4S, 7aR)-4- Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof, for use in treating Fragile X, characterized in that a therapeutically effective amount of said compound or its pharmaceutically acceptable salt is administered to the individual on the basis of the amount of hydroxymethylation compared to a control at one or more of the following positions:
  • the invention includes an mGluR5 antagonist, e.g., (-)-(3aR, 4S, 7aR)-4- Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof, for use in treating Fragile X, characterized in that a therapeutically effective amount of said compound or its pharmaceutically acceptable salt is administered to the individual on the basis of said individual having an amount of hydroxymethylation compared to a control indicative that the subject will respond to treatment with the an mGluR5 antagonist at one or more of the following positions:
  • an mGluR5 antagonist e.g., (-)-(3aR, 4S, 7aR)-4- Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, or a pharmaceutically acceptable salt thereof, for use in treating Fragile X,
  • the invention includes detecting methylation which can be performed using any assay known in the art including methylation-sensitive restriction enzyme digestion combined with PCR or bisulfite DNA modification combined with at least one of: methylation specific PCR (MSP), , probe-based methylation specific PCR or pyrosequencing.
  • MSP methylation specific PCR
  • probe-based methylation specific PCR or pyrosequencing.
  • the invention includes detecting for hydroxymethylation using any assay known in the art.
  • detecting hydroxymethylation can include enriching for 5-hmC- marked DNA by immunoprecipitation and determining the level of enrichment of 5-hmC in the sample.
  • the enrichment level can be determined by, e.g., sequencing, qPCR, or an array.
  • the invention is directed to a diagnostic kit for diagnosing an individual as having Fragile X Syndrome (FXS), including an agent for detecting methylation or hydroxymethylation within one or more regions on chromosome X of the FMRl genomic locus.
  • FXS Fragile X Syndrome
  • the agent can be used to detect DNA hydroxymethylation between 146982000 to 147027400 (herein coordinates based on Human reference genome GRCh37/hgl9), e.g., a predetermined chromosome X region as shown in Table 1, 2 or 3.
  • the predetermined region for hydroxymethylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the predetermined region for hydroxymethylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • the invention is directed to a diagnostic kit for diagnosing an individual as having Fragile X Syndrome (FXS), including an agent for detecting at least two epigenetic biomarkers selected from the group consisting of DNA methylation and hydroxymethylation within a region on chromosome X of the FMRl genomic locus.
  • FXS Fragile X Syndrome
  • Fig. 1 depicts a schematic of the experimental overview of methylated DNA ImmunoPrecipitation ((h)MeDIP) assays combined to a custom DNA microarray platform covering a broad FMRl locus to profile DNA methylation (5mC) and hydroxymethylation (5hmC) in genomic DNA obtained from control and Fragile X syndrome patient samples.
  • (h)MeDIP methylated DNA ImmunoPrecipitation
  • Fig 2 depicts graphs that illustrate the relative enrichment of 5mC and 5hmC (log2 fold enrichment) in control (grey) and FXS (black) fibroblast and PBMC samples (annotated 5mC or 5hmC Fibro or PBMC) over a region covering 79kb on ChrX: 146,971,000-147,050,000).
  • hMeDIP-qPCR individual hydroxymethylation
  • Fig. 4 depicts a graph showing the partial anti-correlation between measured 5mC and 5hmC in the blood of 16 FXS male patients.
  • Fig. 5A depicts a linear regression graph for assay B3 showing the relationship between the methylation (5mC) values (x-axis) and the ABC score (y-axis, the higher the more severe);
  • Fig. 5B shows a table overview of the relationship between the methylation and the ABC score for all assays;
  • Fig 5C depicts a linear regression graph for assay 3F9-G1 showing the relationship between the hydroxymethylation (5hmC) values (x-axis) and the ABC score (y-axis);
  • Fig. 5D shows a table overview of the relationship between hydroxymethylation (5hmC) and the ABC score for all regions assayed.
  • Fig. 6A shows bar graphs of Individual hMeDIP-qPCR assays along the FMRl gene region measuring different levels of 5hmC along the FMRl genomic locus and across a group of control patient (white bars) or clinically treated patient samples and 6B depicts the FMRl genomic locus showing exons, indicated with grey boxes and transcriptional orientation with arrows.
  • the present invention is based, in part, on the finding of novel regions within the FMRl genomic locus that show changes in epigenetic biomarker status compared to a control.
  • the present invention provides critical regions in the FMRl genomic locus that can be used to diagnose subjects suspected of having FXS, or to predict a subject's predisposition thereto.
  • the epigenetic changes in the FMRl gene locus can be used in the selection of particular FXS patients who are likely responders to FXS therapies such as treatment with an mGluR5 antagonist.
  • the sequence of the FMRl gene is known in the art (GenBank L29074 L38501) (Nucleic Acids Res. 2002 Jul 15;30(14):3278-85) (Hum Mol Genet. 2010 Apr 15;19(8): 1618-32. Epub 2010 Jan 29).
  • the FMRl genomic locus covers the FMRl gene sequence and sequences upstream and downstream of the gene sequence.
  • the FMRl genomic locus on chromosome X is between position 146981500 to position 147048300. This region can include a 67 kb region and can include the FMRl coding region, a regionl2 kb upstream of the FMRl coding region and a 16 kb downstream region of the FMRl coding region. All reported chromosomal coordinates as disclosed herein are based on the Human Reference Genome HG19 (Feb. 2009 (GRCh37/hgl9).
  • the epigenetic status in a sample from an individual is detected by determining the presence of one or more epigenetic biomarkers as described herein.
  • the methods of the invention include determining amount or level of the epigenetic biomarker in a sample of interest compared to a control.
  • the epigenetic biomarkers of the invention include DNA methylation (also referred to herein as 5-methylcytosine or 5mC) and hydroxymethylation (also referred to herein as 5- hydroxymethylcytosine or 5hmC).
  • the invention provides predetermined regions, or a portion thereof, (as shown in Table 1, 2 and 3 under "coordinates") within the FMRl genomic locus that can be used in the method of the invention.
  • a portion of a predetermined region includes a region which has a single or group of CpGs.
  • a control as used herein can include a clinically defined sample which has a predefined level of methylation or hydroxymethylation indicative of FXS diagnosis or therapeutic efficiacy, or can be a sample from a healthy subject which is performed at the same time as the methods of the invention. Alternatively, the control can be a statistically validated reference value for use in the methods.
  • Region ID cell type-mark-number
  • kb transcriptional start site
  • 3'UTR 3' untranslated region
  • Table 2 shows the FMRl locus specific methylation and hydroxymethylation assays, the ID used as it corresponds to the figures, the sequence of primers used, the position of each assay along the FMRl regions and the genomic DNA sequence covered by each assay are indicated.
  • the presence of a single epigenetic biomarker such as either methylation or hydroxymethylation can be detected as outlined in Tables 1, 2, or 3, or a combination thereof.
  • the predetermined region for hydroxymethylation can include a region selected from the group consisting of one or more of the following predetermined regions:
  • any two of the regions a-d are assayed, e.g., a and b; or a and c; or b and c, or b and d, etc.
  • any three of the regions a-d are assayed, e.g., a, b and c; or a, c and d; or b, c, and d, etc are assayed.
  • the predetermined region for hydroxymethylation comprises a region selected from the group consisting of one or more of the following predetermined regions:
  • any two of the regions a-f are assayed, e.g., a and b; or a and f; or b and c, or b and d, etc.
  • any three of the regions a-f are assayed, e.g., a, b and c; or a, c and d; or b, c, and d, d, e, and f, etc, are assayed.
  • any four of the regions a-f are assayed, e.g., a, b, c and d; or a, c, d and f; or b, c, d and f; etc, are assayed. Similarly, any five, six or all of the regions a-f are assayed.
  • the presence of at least two or more biomarkers as described herein are detected.
  • the presence of the epigenetic biomarkers as described herein can be determined in any appropriate sample, e.g., blood samples or from other origins (other body fluids, mouth swabs), from an individual of interest.
  • the sample can include a fluid sample such as blood, a cell sample such as PBMCs or buccal cells or fibroblasts, or a tissue sample such as skin or a hair follicle.
  • the methods described herein provide information to enable a health care provider to determine the likelihood that a subject suspected of having FXS, has FXS or a predisposition thereto, or whether an FXS subject will respond to treatment, e.g., using an mGluR5 antagonist.
  • the individual following a positive determination of the relevant epigenetic biomarker(s) in a sample of interest, the individual can be treated with an agent of interest such as an mGluR5 antagonist.
  • an agent of interest such as an mGluR5 antagonist.
  • the individual can be treated with an agent of interest other than an mGluR5 antagonist.
  • DNA methylation includes the epigenetic modification that is catalyzed by DNA cytosine-5- methyltransferases (DNMTs) and occurs at the 5-position (C5) of the cytosine ring within CpG dinucleotides.
  • DNMTs DNA cytosine-5- methyltransferases
  • C5 5-position of the cytosine ring within CpG dinucleotides.
  • changes in methylation include detecting hypermethylation or
  • the extent of FMRl gene methylation in the FMRl gene body can be determined using any method known in the art.
  • the method of the invention includes detecting DNA methylation within the FMRl genomic locus, wherein a change in biomarker status relative to a control is indicative of FXS or the predisposition for FXS.
  • the FMRl gene region of interest to be analyzed for its methylation status according to the present invention can be of any length as long as it includes at least one CpG site, e.g., for DNA methylation can be between position 146993800 to 147048300 and for hydroxymethylation is between 146982000 to 147027400of an FMRl gene sequence.
  • the DNA methylation and hydroxymethylation status of the FMRl gene region to be analyzed is shown in Table 1, 2, and 3.
  • the CGG repeats that are located in the 3'-UTR of the FMRl gene are analyzed for their methylation status and the present invention can be used to complement CGG repeat analysis and enhance diagnosis of FXS in a subject or for use in selecting patients for FXS therapy.
  • Various methods can be used to determine methylation status of an individual of interest such as a qualitative assay such as MSP.
  • Another method useful in the method of the invention is a quantitative assay method such as methylation-sensitive restriction enzyme digestion combined with quantitative PCR, matrix-assisted laser desorption/ionization time-to-flight mass spectrometry (MALDI-TOF- MS), real-time PCR (methyl light).
  • MALDI-TOF- MS matrix-assisted laser desorption/ionization time-to-flight mass spectrometry
  • real-time PCR methyl light
  • the invention is not limited by the types of assays used to assess the extent of methylation of the FMRl gene region in the sample. Indeed, any assay that can be employed to determine the methylation status of a gene can be employed for the purposes of the present invention. Examples of types of assays used to assess the methylation pattern include, but are not limited to:
  • methylation-sensitive restriction enzyme digestion combined with at least one of: hybridization, quantitative PCR, restriction landmark genomic scanning (RLGS), or array-based profiling of reference-independent methylation status (aPRIMEs);
  • RNA modification combined with at least one of: methylation specific PCR (MS-PCR), quantitative methylation specific PCR (qMS-PCR), probe-based methylation specific PCR, pyrosequencing, cloning/sequencing, MS-nested PCR, quantitative analysis of methylated alleles (QUAMA), heavy methyl detection, methylation-sensitive high resolution melting (MS-HRM), methyl-binding (MB)- PCR, PCR and deoxyribonucleoside monophosphate (dNMP) analysis, or methylation-dependent fragment separation (MDFS);
  • MS-PCR methylation specific PCR
  • qMS-PCR quantitative methylation specific PCR
  • probe-based methylation specific PCR probe-based methylation specific PCR
  • pyrosequencing pyrosequencing
  • cloning/sequencing MS-nested PCR
  • quantitative analysis of methylated alleles QUAMA
  • heavy methyl detection methylation-sensitive high resolution melting (MS-HRM
  • the extent of methylation can be determined using Methylation Specific PCR (MSP).
  • MSP is a bisulfite conversion based PCR technique that can be used to determine DNA CpG
  • MSP involves the initial modification of DNA by sodium bisulfite which converts all unmethylated, but not methylated, cytosines to uracil.
  • the DNA is then amplified with two pairs of primers specific for methylated DNA and unmethylated DNA, respectively, and the methylation status determined.
  • the primers typically include at least two CpG sites.
  • the MSP methods are described in U.S. Pat. No. 5,786,146; U.S. Pat. No. 6,017,704; U.S. Pat. No. 6,200,756; and U.S. Pat. No. 6,265,171; the entire contents of each of which is incorporated herein by reference.
  • an individual would be assigned as an mGluR5 responder when the methylated FMR1 is detected by the primers specific for methylated DNA and unmethylated FMR1 is not detected by the primers specific for unmethylated DNA in the region of interest of the FMR1 gene.
  • the extent of methylation can be determined using a method that includes an amplification process such as quantitative PCR (qPCR) in the FMR1 gene region of interest.
  • qPCR quantitative PCR
  • the qPCR method described in WO2011/137206 can be used.
  • the method described in WO2011/137206 is incorporated herein by reference.
  • Various other different qPCR methods which detect methylation are known in the art and include HeavyMethyl or Methylight.
  • the FMR1 gene region is initially modificated by sodium bisulfite.
  • the DNA is then contacted with non-extendable oligonucleotide blockers that provide specificity by binding to bisulfite-treated DNA in a methylation-specific manner.
  • the DNA is then contacted with a primer set that has binding sites that overlap with non-extendable oligonucleotide blockers.
  • the primer cannot bind and therefore no amplicon is generated.
  • the primers can bind and generate an amplicon (Cottrell et al. Nucleic Acids Res. 2004; 32(1), 2004).
  • the FMR1 gene region of interest is initially modified by sodium bisulfite.
  • the gene region is then amplified using PCR primers that hybridize to regions containing no CpG dinucleotides.
  • fluorescent probe detection can indicate methylation status of sequences where the probes hybridize.
  • Methods for detecting methylation of a region of interest by cutting the DNA with a methylation-sensitive restriction enzyme and subsequently selectively identifying and/or analyzing the cut or uncut DNA are known in the art.
  • the method can encompass amplifying intact DNA after restriction enzyme digestion see, e.g., U.S. patent application Ser. Nos. 10/971,986; 11/071,013; and 10/971,339.
  • the method of the invention includes digesting the FMR1 gene region of interest with a methylation sensitive restriction enzyme and amplifying up the region of interest.
  • the methylation status of the DNA can be determined by detecting for the presence of an amplifiable product. Only DNA that was not cleaved by the restriction enzyme will be amplified.
  • a methylation sensitive restriction enzyme can be for example, McrBC, which includes CG as part of its recognition site and can cleave when the C is methylated.
  • McrBC which includes CG as part of its recognition site and can cleave when the C is methylated.
  • the sample can be contacted with a restriction enzyme which includes CG as part of its recognition site and can cleave only when the C is unmethylated.
  • the desired FMR1 region can be amplified by real-time PCR using a
  • the probe for detecting nucleic acid sequence typically has a fluorescent reporter or fluorophore such as 6-carboxyfluorescein (FAM) and
  • TET tetrachlorofluorescin
  • TAMRA tetramethylrhodamine
  • BHQ black hole quencher
  • the methods described above can be used with a methylation analyzer.
  • the method includes determining the extent of FMR1 methylation in the sample, transforming the results into a computer readable form and applying a mathematical algorithm to classify the results into a classification group, i.e., diagnosis of FXS.
  • the methods described above include control samples such as samples that are fully methylated and samples that are partially methylated. DNA purified from fragile X patient's B- lymphocytes (Camden, NJ) can be used to generate appropriate controls or clinical samples which are already determined to have a particular methylation status can be used.
  • the methods described above include control samples.
  • Such samples are readily available in the art or can be commercially purchased from, e.g., ATCC (American Type Culture Collection (ATCC), The National Institute for Biological Standards and Control (NIBSC) or Coriell institute for medical research.
  • ATCC American Type Culture Collection
  • NBISC National Institute for Biological Standards and Control
  • the control can either be run simultaneously with the test sample or can be represented as a predetermined value based on the technology used to determine the methylation status of the sample.
  • the predetermined value is a Delta Ct value which is obtained using quantitative PCR (as described herein).
  • the oligonucleotides of the invention can include any oligonucleotide as disclosed in Table 1 , 2 and 3 and any other oligonucleotide that can be used to detect a predetermined region as described herein.
  • the oligonucleotides of the invention include variants of the sequences or sequences that are
  • substantially similar to the oligonucleotides of the invention Variants include sequences that are altered by one or more bases, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 but can still anneal to the specific locations on the FMRl sequence of interest.
  • the term "substantially” when used in relation to annealing or hybridisation means that the oligonucleotide or probe nucleic acid sequence should be sufficiently complementary to hybridise or anneal to its respective nucleic acid. As used herein, the term
  • hybridisation refers to the process by which a strand of nucleic acid joins with a complementary strand.
  • the oligonucleotide is between 14-30 bases. In another example, the oligonucleotide is between 18-30.
  • Oligonucleotides may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • the invention further includes detecting hydroxymethylation in the FMRl locus, wherein the amountof hydroxymethylation (5-hmC) relative to a control is indicative of FXS or the predisposition for FXS, or can be used to stratify patients receiving an mGluR5 antagonist as described herein.
  • 5-hmC is a hydroxylated and methylated form of cytosine.
  • the FMRl gene region of interest to be analyzed for its 5-hmC status according to the present invention can be of any length. Examples of regions are shown in Tables 1 and 2.
  • 5-hmC in a sample of interest can be detected by first enriching for 5-hmC-marked DNA by immunoprecipitation and then quantifying for the presence of 5-hmC by either sequencing, qPCR, or arrays.
  • 5-hmC can be detected by using either HPLC, LC/MS or single molecule real-time DNA sequencing (SMRT sequencing)
  • hydroxymethylation can be measured as described herein using hMeDIP- qPCR with selected primers (see Tables land 2).
  • the present invention of detecting hydroxymethylation as described herein can be used in combination with the standard methylation clinical diagnostic assays such as detecting methylation at the FMRl promoter (e.g., SEQ ID NO: l or a portion thereof) or using the assay as described in WO2011/137206, which is incorporated in its entirety herein by reference in order to stratify patients who are more likely to respond to an mGluR5 antagonist.
  • the FMRl gene promoter is analyzed for its methylation status.
  • the FMRl promoter region being analyzed is the full length promoter region.
  • a portion of the FMRl promoter region is analyzed for its methylation status.
  • the FMRl gene promoter region has the nucleotide sequence of SEQ ID NO: 67 (shown below; CpG islands are shown in bold and underlined).
  • the present invention can be used to determine if an individual will respond to FXS drug therapies.
  • the invention can be used to determine if an individual having FXS are likely to respond to treatment with an FXS treating agent such as an Metabotropic Glutamate Receptor 5 (mGluR5) antagonist.
  • an mGluR5 responder is an individual having FXS who is likely following therapeutic treatment with an mGluR5 antagonist to show improved behavioral symptoms as assessed using the Aberrant Behavior Checklist - Community Edition (ABC-C) measure of behavior (Bihm et al., Am. J. Ment Retard 96:209-211).
  • the ABC-C measurement looks at various behaviors including stereotypic behavior, hyperactivity, inappropriate speech, and restricted interests.
  • An individual who shows a decrease in ABC-C scores following treatment with an mGluR5 antagonist is classified as an mGluR5 responder.
  • the behavioral symptoms may also be assessed by other methods, such as Clinical Global Impression (CGI) scale, Social Responsiveness Scale (SRS), or Repetitive Behavior Scale - Revised (RBS-R). Individuals showing an improvement according to these tests will also be determined to be an mGluR5 responder.
  • CGI Clinical Global Impression
  • SRS Social Responsiveness Scale
  • RBS-R Repetitive Behavior Scale - Revised
  • an mGluR5 non-responder is an individual having FXS who is unlikely following therapeutic treatment with an mGluR5 antagonist to show improved behavioral symptoms as assessed using the Aberrant Behavior Checklist - Community Edition (ABC-C) measure of behavior (Bihm et al, Am. J. Ment Retard 96:209-211). Such an individual should based on a negative finding using the methods of the invention, be administered a compound other than an mGluR5 antagonist.
  • mGluR5 antagonists include eptidomimetics, proteins, peptides, nucleic acids, small molecules, or other drug candidates.
  • An example of an mGluR5 antagonist is (-)-(3aR, 4S, 7aR)-4- Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester.
  • the mGluR5 antagonist, (- )-(3aR, 4S, 7aR)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester, as well as methods of making the same, are disclosed in U.S.
  • mGLUR5 antagonists such as those disclosed in U.S. Patent No. 7,348,353 are contemplated for use in the methods of the present invention.
  • the method can be used to select those individuals having FXS who are likely to respond to therapy with an mGluR5 antagonist based on the amount of methylation or
  • the mGluR5 antagonist is a compound of the formula (I)
  • R represents optionally substituted alkyl or optionally substituted benzyl
  • R 2 represents hydrogen (H), optionally substituted alkyl or optionally substituted benzyl; or
  • R 1 and R 2 form together with the nitrogen atom to which they are attached an optionally substituted heterocycle with less than 14 ring atoms;
  • R 3 represents halogen, alkyl, alkoxy, alkylamino or dialkylamino
  • R 4 represents hydroxy (OH), halogen, alkyl or alkoxy
  • Q represents CH, CR 4 or N
  • Y represents CH, CR 4 or N
  • W represents CH, CR 4 or N
  • X represents CH or N
  • Y represents CH, CR 3 or N
  • Z represents CH 2 , NH or O
  • the mGluR5 antagonist is a compound of the formula (II), wherein a compound of the formula (II) is a compound of formula (I) in which at least one of Q, V and W is N; in free base or acid addition salt form.
  • mGluR5 antagonist is a compound of the formula (III), wherein the compound of formula (III) is a compound of formula (II) in which Y is CR 3 ; in free base or acid addition salt form.
  • X preferably represents CH.
  • Y preferably represents CH or CR 3 , wherein R 3 preferably represents halogen, particular preferably chloro.
  • Z preferably represents NH.
  • R 3 preferably represents fluoro, chloro, Ci_ 4 alkyl, e.g. methyl.
  • R 3 particularly preferably represents chloro.
  • R 1 and R 2 form together with the nitrogen atom to which they are attached form an
  • R 1 and R 2 preferably form together with the nitrogen atom to which they are attached an unsubstituted, a single or twofold substituted heterocycle selected from the group
  • substituents being selected from the group consisting of fluoro, chloro, methyl, ethyl, propyl, butyl, trifluoromethyl, fluoropropyl and difluoropropyl.
  • R 1 and R 2 preferably represent, independently from each other, Ci-C 4 alkyl or benzyl,
  • Ci-C 4 alkoxy or halogen optionally substituted by Ci-C 4 alkoxy or halogen.
  • radical definitions apply both to the end products of the formulae (I), (II) and (III) and also, correspondingly, to the starting materials or intermediates required in each case for the preparation. These radical definitions can be combined with one another at will, i.e. including combinations between the given preferred ranges. Further, individual definitions may not apply. Preference according to the invention is given to compounds of the formulae (I), (II) and (III) which contain a combination of the meanings mentioned above as being preferred.
  • R represents halogen, preferably chloro, and the other substituents have the meaning given in this specification.
  • the mGluR5 antagonist is a compound of the formula (IV):
  • n 0 or 1
  • n 0 or 1
  • A is hydroxy
  • X is hydrogen and
  • Y is hydrogen, or
  • A forms a single bond with X or with Y;
  • Ro is hydrogen, (Ci_ 4 )alkyl, (Ci_ 4 )alkoxy, trifluoromethyl, halogen, cyano, nitro, -COORi wherein Ri is (Ci_ 4 )alkyl or -COR 2 wherein R 2 is hydrogen or (Ci_ 4 )alkyl, and
  • R is -COR 3 , -COOR 3 , -CONR 4 R 5 or -S0 2 Re, wherein R 3 is (Ci_ 4 )alkyl, (C 3 - 7 )cycloalkyl or optionally substituted phenyl, 2-pyridyl or 2-thienyl; R 4 and R 5 , independently, are hydrogen or (Ci_ 4 )alkyl; and 5 is (Ci_ 4 )alkyl, (C 3 _ 7 )cycloalkyl or optionally substituted phenyl, R is hydrogen or (Ci_ 4 )alkyl and R" is hydrogen or (Ci_ 4 )alkyl, or
  • R and R" together form a group -CH 2 -(CH 2 ) m - wherein m is 0, 1 or 2, in which case one of n and m is different from 0,
  • Ro is different from hydrogen, trifluoromethyl and methoxy when n is 0,
  • A is hydroxy, X and Y are both hydrogen, R is COOEt and R' and R" together form a group -' (CH 2 )2-, in free base or acid addition salt form.
  • Exemplary compounds of formula (IV) include:
  • the mGluR modulator is a compound of the formula (V):
  • R represents hydrogen or alkyl
  • R 2 represents an unsubstituted or substituted heterocycle
  • R 2 represents an unsubstituted or substituted aryl
  • R 3 represents alkyl or halogen
  • X represents a single bond or an alkandiyl-group, optionally interrupted by one or more oxygen atoms or carbonyl groups or carbonyloxy groups in free base or acid addition salt form.
  • Exemplary compounds of formula (V) include:
  • Furan-3-carboxylic acid [(1 S,3S)-3-(3-chloro-phenylethynyl)-3-hydroxy-cyclohexyl]-amide
  • Furan-2-carboxylic acid [(1 R,3R)-3-(3-chloro-phenylethynyl)-3-hydroxy-cyclohexyl]-amide
  • Furan-2-carboxylic acid [(1 S,3S)-3-(3-chloro-phenylethynyl)-3-hydroxy-cyclohexyl]-amide
  • Furan-3-carboxylic acid ((1 R,3R)-3-hydroxy-3-m-tolylethynyl-cyclohexyl)-amide
  • Furan-3-carboxylic acid ((1 S,3S)-3-hydroxy-3-m-tolylethynyl-cyclohexyl)-amide
  • Furan-3-carboxylic acid (( ⁇ )-(l R.3R)-3-hydroxy-3-m-tolylethynyl-cyclohexyl)-amide
  • Furan-2-carboxylic acid ((1 R,3R)-3-hydroxy-3-m-tolylethynyl-cyclohexyl)-amide
  • Furan-2-carboxylic acid ((1 S,3S)-3-hydroxy-3-m-tolylethynyl-cyclohexyl)-amide
  • Furan-2-carboxylic acid (( ⁇ )-(l R,3R)-3-hydroxy-3-m-tolylethynyl-cyclohexyl)-amide
  • Furan-3-carboxylic acid [(1 S,3S)-3-(3-fluoro-phenylethynyl)-3-hydroxy-cyclohexyl]-amide
  • Furan-2-carboxylic acid [(1 R,3R)-3-(3-fluoro-phenylethynyl)-3-hydroxy-cyclohexyl]-amide
  • Furan-2-carboxylic acid [(1 S,3S)-3-(3-fluoro-phenylethynyl)-3-hydroxy-cyclohexyl]-amide
  • 6-Chloro-pyridine-2-carboxylic acid [( IS, 3 S)-3 -(3 -chloro-phenylethynyl)-3 -hydroxy-cyclohexyl] -amide 5 -Chloro-1 -methyl- 1 H-pyrrole-2-carboxylic acid [(1 R,3R)-3-(3-chloro-phenylethynyl)-3-hydroxy- cyclohexyl]-amide
  • the mGluR modulator is a compound of the formula (VI)
  • R 1 represents hydrogen or alkyl
  • R R 2 represents an unbubstituted or substituted heterocycle
  • R 2 represents an unsubstituted or substituted aryl
  • R 3 represents alkyl or halogen
  • mGluR5 antagonists include compounds of the formula (I) as defined in WO
  • the methods described herein can be utilized as a diagnostic assay to identify those subjects having Fragile X Syndrome or a predisposition thereto.
  • the methods described herein can be used to identify subjects who are likely to respond to an FXS therapy such as an mGluR5 antagonist or can be used as a prognostic assay to identify subjects who are at risk of developing Fragile X Syndrome and who would benefit from receiving an FXS therapy such as an mGluR5 antagonist.
  • Prognostic assays can be used for predictive purposes or prophylactic purposes to treat an individual who is at risk of developing FXS.
  • the methods of the invention include determining the amount of DNA methylation and/or
  • hydroxymethylation in a predetermined region as described in Table 1, 2, or 3 in a test subject and comparing the amount determined to a control.
  • a diagnosis as to whether that subject has FXS or a predisposition thereto, or whether that subject will respond to particular therapy can be made.
  • control for use in the methods for detecting methylation or hydroxymethylation as described herein can be any appropriate control or set of controls.
  • the control can be a clinical sample which has a defined amount of methylation indicative of FXS or predisposition thereto, or positive response to an mGluR5 therapy.
  • controls can be fully methylated or partially methylated, or can be from a subject which does not have FXS (healthy subject).
  • Clinical samples taken from individuals that are fully methylated (or more than 95% methylated), or are fully methylated and mGluR5 responders, can serve as positive controls and samples that are partially methylated or from subjects not having FXS can serve as negative controls.
  • the control is a sample from a subject that does not have FXS.
  • an increase in methylation in a test sample compared to the control is indicative that the subject has FXS or will likely respond to an mGluR5 antagonist.
  • the control is a threshold value which has been previously determined from clinically relevant samples (such as samples positive for FXS or positive for response to an mGluR5 antagonist) at one or more of the predetermined regions described herein. The amount of methylation in the test sample can then be compared against the threshold value and a diagnostic or appropriate therapy determination can be made.
  • a control can be a sample or value generated from a clinical sample that has already been determined to have a particular clinically relevant hydroxymethylation status.
  • the control can be generated from clinical samples where the subject is an mGluR5 responder and has a defined hydroxymethylation status. Samples taken from individuals that are an mGluR5 responder can serve as positive controls and samples that are from subjects not having FXS or non responders can serve as negative controls. In one example, where the control is from a subject that does not have FXS, a decrease in hydroxymethylation in a test sample compared to the control is indicative that the subject will respond to an mGluR5 antagonist. In another example, the control is a threshold value which has been determined by measuring the relative level of hydroxymethylation for one or more of the predetermined regions in a clinically relevant sample. The amount of hydroxymethylation in the test samples can then be compared against the threshold value and a diagnostic or therapy determinations can be made.
  • the control can either be run simultaneously with the test sample or can be represented as a
  • the predetermined value based on the technology used to determine the methylation or hydroxymethylation status of the sample.
  • the predetermined value is a Delta Ct value which is obtained using quantitative PCR.
  • the amount of DNA methylation and/or hydroxymethylation in a predetermined region as described in Table 1, 2 and 3 compared to a control can be indicative as to whether that subject will respond to an FXS therapy such as an mGluR5 responder.
  • the method of the invention can be used as a prognostic assay to determine whether a subject should be administered an FXS therapy such as an mGluR5 antagonist so as to prevent the onset of Fragile X syndrome or to reduce the severity of Fragile X syndrome.
  • an individual can be determined to be at risk of developing FXS using any of the standard methods known in the art such as detecting CGG repeats, evaluating the family history of that individual or using the method described herein.
  • an individual is further evaluated for the presence of any one or more of the above described epigenetic biomarkers.
  • the presence of one or more of the biomarkers described herein can be used to indicate that that individual should be administered an mGluR5 antagonist so as to prevent the onset of Fragile X syndrome or to reduce the severity of Fragile X syndrome.
  • newborn infants determined to be at risk of developing FXS should be monitored for the presence of one or more of the biomarkers described herein so as to prevent the onset of Fragile X syndrome or to reduce the severity of Fragile X syndrome.
  • the use of the present method to intervene early will maximize the therapeutic benefits of mGluR5.
  • the prognostic assay described herein can also be used in any individual who exhibits CGG repeat length expansion in the FMRl gene. If an individual is determined based on the methods described herein to be an individual who will respond clinically to an mGluR5 antagonist, an mGluR5 antagonist will be administered to the individual. In general, a daily dosage in the range from about 5 to 1500 mg, preferably about 10 to about 1000 mg of the compound is conveniently administered to an individual having FXS. In one example, a daily dosage of 10 mg, 25 mg or 100 mg will be administered to the individual having FXS.
  • the epigenetic biomarkers described herein can be used to further characterize subjects having FXS who have varying amounts of methylation associated with the FMRl promoter region.
  • the DNA methylation and/or hydroxymethylation profile in the broader FMRl genomic locus as detailed in Table 1 further information about subjects having a fully methylated promoter or a partially methylated promoter can be obtained which will be useful for determining the best options with respect to therapeutic treatments.
  • kits for detecting the status of methylation of the FMRl genomic locus can be used to determine if a subject has FXS or if a subject having FXS is likely to respond to treatment with an FXS therapy such as mGluR5 antagonist.
  • the kit can comprise a labeled compound or agent capable of detecting the status of DNA methylation and/or hydroxymethylation in the broader FMRl genomic locus.
  • the kit can also include primers that can be used to determine the status of DNA methylation and/or hydroxymethylation in the broader genomic locus , e.g., as disclosed in Table 1, 2, and 3 and an appropriate control sample.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container, and all of the various containers are within a single package along with instructions for it's use.
  • the result can be cast in a transmittable form of information that can be communicated or transmitted to other researchers or physicians or genetic counselors or patients.
  • a form can vary and can be tangible or intangible.
  • the result can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms.
  • images of gel electrophoresis of PCR products can be used in explaining the results or graphs of qPCR can be used.
  • These statements and visual forms can be recorded on a tangible media such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible media, e.g., an electronic media in the form of email or website on internet or intranet.
  • the result can also be recorded in a sound form and transmitted through any suitable media, e.g., analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like. All such forms (tangible and intangible) would constitute a "transmittable form of information".
  • the information and data on a test result can be produced anywhere in the world and transmitted to a different location.
  • the present disclosure also encompasses a method for producing a transmittable form of information containing data on the status of methylation and/or hydroxymethylation in the broader FMRl genomic locus in a test sample. This form of information is useful for diagnosis of FXS or predisposition thereto, or to select a test patient who will respond to FXS treatment such as mGluR5 treatment, or for selectively treating a patient based upon that information.
  • Example 1 Methylome and hydroxymethylome mapping of the broader FMRl genomic locus was performed. Chromatin profiling assays (Methylated DNA Immunoprecipitation (MeDIP)) combined to a DNA microarray covering a broad genomic region encompassing the FMRl gene sequence. Broader epigenomic profiling of the FMRl locus of FXS fibroblasts.
  • 5-Methylcytosine (5mC) and 5-Hydroxymethylcytosine (5hmC) MeDIP assays using highly specific anti-5mC and anti-5hmC antibodies were carried out on genomic DNA from 3 FXS fibroblasts lines and 2 control lines. The same assays were also run on genomic DNA obtained from 5 control PBMC samples and up to 11 FXS samples (Fig. 1).
  • MeDIP enrichment was validated using qPCR at candidate loci previously identified as being marked by the indicated epigenetic modifications. MeDIP enriched input and IP fractions were labeled and applied onto a custom designed array covering the entire FMRl genomic region on chromosome X (ChrX: 146,911,760 - 147,159,387: 82 kb of upstream and 126kb of downstream regions flanking the 40kb FMRl gene. This overall experimental design generated 40 independent microarray datasets, enabling the first epigenomic profiling of the broader FMRl locus in control and FXS cells across a variety of control and FXS individuals (fibroblasts and PBMCs).
  • Analyses of epigenome landscape using genome visualization tools detected the expected DNA hypermethylation in the region surrounding the FMRl promoter and transcriptional start site, including the upstream (5') part of FMRl intron 1 (TSS -lkb/+2.5kb, referred as to "Fibro-5mC-C” and “Fibro- 5mC-D” regions in Table 3) in FXS fibroblasts.
  • MeDIP profiles from single cell lines showed that methylation patterns were consistent across fibroblast lines (data not shown).
  • PCR primers were designed and quantitative PCRs run on MeDIP and input material from FXS and control cells, confirming decreased methylation in the newly identified regions of FMRl methylation changes in FXS fibroblasts, while illustrating the robust TSS region hypermethylation.
  • additional control regions were investigated and did not show change in methylation across cell lines (GAPDH, APRT: constitutive ly hypomethylated and HI 9, NNAT, IGF2R: constitutively hypermethylated) (data not shown).
  • Table 3 summarizes all region of 5mC change in fibroblasts with functional location, and chromosomal coordinates.
  • DNA methylation changes are associated to hydroxymethylation perturbations at specific locations along the FMRl locus in FXS fibroblasts.
  • PCR analyses of relative 5mC and 5hmC enrichments in the TSS region identified, compared to control PBMC samples, varying levels of increased methylation and decreased hydroxymethylation across the FXS PBMC samples.
  • PCR data and array data were found consistent, with in particular patient samples #8 and #12 showing highest levels of 5hmC, patients #8, #15 and #17, intermediate levels of 5hmC and patients #9, #10, #11 and #13 the lowest levels of 5hmC. Comparing DNA methylation and hydroxymethylation in selected patients showed correlation between 5mC and 5hmC (compare patient #9, high 5mC, low 5hmC and patient #12, low 5mC and high 5hmC).
  • Hypermethylation at the region surrounding the TSS is accompanied by a strong hypermethylation peak within the first intron of FMRl, in a region with lower CpG density as compared to FMRl promoter, as previously reported by Golder et al. (Godler et al., 2010 Hum Mol Genet. 2010 Apr 15;19(8): 1618-32).
  • Increased methylation upstream and downstream of the CGG repeats participate to the suppression of FMRl gene expression and may be a consequence of the repeat expansion and associated epigenetic switch.
  • the other regions of methylation changes in fibroblast cells show hypomethylation in FXS cells (region 5' and gene body).
  • the novel pyrosequencing assays developed herein provide an enhanced method for molecular characterization of FXS patient samples.
  • hMeDIP-qPCR assays at 6 regions of differential 5hmC encompassing the FMRl genomic region (Table 1, Table 2, Figure 3B) were developed.
  • Assay 3H3-4 lOkb upstream of FMRl transcriptional start site (TSS) localizes with the transcriptional start site of a 534bp non-coding RNA entitled L29074.3 (ENST00000597190) of unknown function.
  • Assays 3C1-2, and 213-4 are localized in the vicinity of FMR1 promoter region, upstream of the TSS.
  • Assays 3C7-9 and 3E8-9 are localized within the intron 1 of FMR1 and assay 3F9- Gl is localized 31.4 kb downstream of FMR1 TSS, within alternative-splicing rich regions of FMR1.
  • the deployment of these assays on 5 control DNA samples from healthy patient PBMCs confirmed our previous MeDIP-array data (Figure 2) with strong enrichment of the 5hmC mark in the promoter and intron 1 areas of the FMR1 locus.
  • the relative enrichment of 5hmC across individual control samples was highly consistent (Figure 3B, white bars).
  • the graphs illustrate the relative enrichment of 5mC (top) and 5hmC (bottom) (log2 fold enrichment) in 5 control PBMC (grey) and 1 illustrative FXS (black) PBMC samples over a region covering 79kb on ChrX: 146,971,000-147,050,000).
  • the position of the FMR1 gene and non-coding RNA L29074.3 is indicated. Exons are indicated with grey boxes and transcriptional orientation with arrows.
  • TSS Transcriptional Start Site.
  • the regions of change of methylation and hydroxymethylation are indicated with dashed lines and refer to the regions listed in Table 1.
  • Example 3 Combined 5mC and 5hmC measurements enhance molecular and functional characterization of FXS patient samples
  • Figure 4 illustrates the relationship between 5mC and 5hmC at two independent regions of FMRl, showing a significant correlation between 5mC levels at the peri-promoter region of FMRl (commercial Qiagen assay HsFMRl) and 5hmC levels within the intron 1 of FMRl, 8kb downstream of the TSS (assay 3E8-9).
  • figure 4 shows the relationship between the methylation (5mC) and the hydroxymethylation (5hmC) values is shown using simple linear regression analysis (see methods).
  • the coefficient of determination, denoted R 2 and the p-value for the data correlation are indicated.
  • the mRNA expression levels are expressed by the color intensity, the darker the more FMRl mRNA measured.
  • Example 4 FMRl methylation and hydroxymethylation is significantly correlated with ABC scores in FXS male patients.
  • the relationship between the methylation (5mC) values (x-axis) and the ABC score (y-axis, the higher the more severe) is indicated in the linear regression graph for assay B3 (5A) and for all assays in the table overview (5B).
  • the relationship between the hydroxymethylation (5hmC) values (x- axis) and the ABC score (y-axis) is indicated in the linear regression graph for assay 3F9-G1 (5C) and for all assays in the table overview (5D). Simple linear regression analysis were used for these analyses (see methods).
  • the middle panel graphs illustrate the relative enrichment of 5mC (top) and 5hmC (bottom) (log2 fold enrichment) as in Figure 1 and 3.
  • the data confirms the utility of 5mC to predict FXS disease severity and suggests for the first time that 5hmC measurements within the FMRl locus may be equally indicative of disease severity. Combined assessment of 5mC and 5hmC within selected regions of the FMRl locus may thus significantly enhance clinical diagnostics for Fragile X syndrome.
  • the Partial (PM) or Full (FM) methylation status of the FXS samples 1 to 5 is indicated as well as each patient's response to (-)-(3aR, 4S, 7aR)-4-Hydroxy- 4-m-tolylethynyl-octahydro-indole-l-carboxylic acid methyl ester (Responder, R; Non-responder, NR).
  • Responder, R; Non-responder, NR Non-responder, NR.
  • GM04858 fibroblasts from a 4 year old fragile X patient GM07072 fetal lung fibroblasts from 22 week old fetus with a fragile mutation and GM09497 fibroblasts from a 28 year old fragile X patient, from Coriell Institute for Medical Research were grown in D-MEM supplemented with 15% FBS, penicillin/streptomycin, 2-mercaptoethanol (O. lmM) and sodium pyruvate.
  • the cell lines used as controls, BJ1 fibroblasts and MG63 osteosarcoma cell line from ATCC were cultured in the same condition.
  • PBMC were purified from blood using an 8 mL capacity PBMC separator tubes (BD Vacutainer CPT, BD).
  • Genomic DNA from fibroblasts and control cell lines was prepared by overnight Proteinase K (pK) treatment in lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 100 mM NaCl, 0.5% SDS), phenol-chloroform extraction, ethanol precipitation and RNaseA digestion. Genomic DNA was sonicated (Bioruptor, Diagenode) to produce random fragments ranging in size from 300 to 1,000 bp and 4 ⁇ g of fragmented DNA was used for a standard hMeDIP assay.
  • DNA was denatured for 10 min at 95°C and immunoprecipitated for 3 hrs at 4°C with 15 ⁇ of monoclonal antibody against 5- methylcytidine (BI-MECY-1000, Eurogentec) (MeDIP) or with ⁇ of a rabbit polyclonal antibody against 5-hydroxymethylcytosine (#39769, active Motif) (hMeDIP) in a final volume of 500 ⁇ IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100).
  • IP buffer 10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100.
  • the mixture was incubated with 40 ⁇ magnetic beads (MeDIP: Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen) for 2 hrs at 4°C / hMeDIP: Dynabeads Protein G (#100.03D, Invitrogen) for lhr at 4°C) and washed three times with 1ml of IP buffer. Beads were subsequently treated with proteinase K for 3 hrs at 50°C and the methylated DNA recovered by phenol-chloroform extraction followed by ethanol precipitation.
  • MeDIP Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen) for 2 hrs at 4°C / hMeDIP: Dynabeads Protein G (#100.03D, Invitrogen) for lhr at 4°C
  • Table 5 List of primers used for methylation profiling at the FMR1 genomic locus
  • lug of genomic DNA was sonicated (Bioruptor, Diagenode) to produce random fragments ranging in size from 300 to 1,000 bp and 500ng of fragmented DNA was used for the hMeDIP assay.
  • DNA was denatured for 10 min at 95°C and immunoprecipitated for 3 hrs at 4°C with 400ng of a rabbit polyclonal antibody against 5-hydroxymethylcytosine (#39769, active Motif) (hMeDIP) in a final volume of 200 ul IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100).
  • the temperature profile of the cycles was DNA polymerase activation at 95°C for 15min, denaturation at 95°C for 30sec, annealing at 61°C for 30 sec, and extension at 72°C for 1 min for the first cycle.
  • the annealing temperature was decreased by 0.5°C per cycle.
  • 36 cycles of amplification were performed at 53°C, the final annealing temperature.
  • Biotinylated PCR product were then purified and immobilized onto streptavidin-coated Sepharose beads (GE Healthcare). Pyrosequencing was performed on the PyroMark Q96 MD (Biotage/Qiagen) following the manufacturer's instructions. Pyro QCpG 1.0.9 (Bioatage/Qiagen) was used to quantify DNA methylation at single CpGs. Data analyses Statistical analyses
  • Percentage of methylation per CpG obtained by pyrosequencing was summarized by averaging the value of all CpGs per assay, 0% being unmethylated and 100% fully methylated. 5hmC MeDIP-qPCR data were first normalized using the efficacy of each qPCR assay. The ratio IP/Input was calculated and is expressed as percent of Input, with higher values representing a stronger enrichment for the measured mark, 5hmC. The relationship between the methylation and the hydroxymethylation values to the clinical score (Aberrant Behavior Checklist (ABC C) score (Aman et al, 1995, supra)) was assessed via simple linear regression analysis. The coefficient of determination, denoted R 2 and the p-value for the clinical vs methylation data correlation are indicated. All analyses were conducted with TIBCO Spotfire 4.0.2.

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Abstract

L'invention concerne de nouvelles régions à l'intérieur du locus génomique FMR1 qui peuvent servir en tant que biomarqueurs pour diagnostiquer le Syndrome de l'X Fragile (FXS), ou une prédisposition pour celui-ci, ou pour déterminer la sensibilité d'un individu atteint de FXS vis-à-vis d'un traitement par un agent thérapeutique pour FXS.
PCT/EP2013/054542 2012-03-08 2013-03-06 Marqueurs de prédiction utiles dans le diagnostic et le traitement du syndrome de l'x fragile (fxs) WO2013131981A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114807360A (zh) * 2022-06-27 2022-07-29 北京贝瑞和康生物技术有限公司 检测脆性x综合征突变的方法和试剂盒
CN114807360B (zh) * 2022-06-27 2022-09-02 北京贝瑞和康生物技术有限公司 检测脆性x综合征突变的方法和试剂盒

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