Treatment and diagnosis of epigenetic disorders and conditions

Abstract

The present disclosure relates generally to the field of epigenetics and in particular epigenetic profiles associated with a pathological condition. The present specification teaches screening of individuals and populations for epigenetic profiles associated with a pathological condition. The epigenetic profiles can be from an intron, an intron/exon boundary or a splicing region. Epigenetic profiles are disclosed from the following sites in the FMR locus: FREES, intron 2 of FMR1, the genomic FREE2 region as a whole or specific FREE2 fragments including FREE2 (D) or FREE2 (E). Kits and diagnostic assays are also taught herein as are computer programs to monitor changes in epigenetic patterns and profiles. Further enabled herein is a method for screening for agents which can reduce or mask the adverse effects of epigenetic modification and the use of these agents in therapy and prophylaxis.

1 . A method for identifying a trinucleotide expansion disorder in a mammalian subject including a human, said method comprising screening for a change relative to a healthy control in the extent of epigenetic modification within (i) two or more of (a) an intron; (b) an intron/exon boundary; and/or (c) a splicing region; and/or (ii) approximately one seventh or greater of an intron including an intron/exon boundary and/or a splicing region of the FMR genetic locus; wherein a change in extent of epigenetic modification relative to the control is indicative of the presence or severity of the trinucleotide expansion disorder or a propensity to develop same wherein the intron, intron/exon boundary and/or splicing region is selected from the group consisting of:
 (i) Fragile X-related Epigenetic Element 3 in FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:1 or a homolog thereof or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:1 or which hybridizes to SEQ ID NO:1 or its complementary form under medium stringency conditions;   (ii) intron 2 of FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:2 or a homolog thereof or a portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:2 or which hybridizes to SEQ ID NO:2 or its complementary form under medium stringency conditions; and   (iii) genomic FREE2 region as a whole or specific fragments of FREE2 including FREE2 (D), FREE2 (E) or FREES comprising the nucleotide sequence set forth in SEQ ID NO:48 or 49 or 47, respectively or a homolog or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:48 or 49 or 47 or which hybridizes to SEQ ID NO:48 or 49 or 47 or its complementary form under medium stringency conditions.   
     
     
         2 . The method of  claim 1  wherein the trinucleotide expansion disorder is selected from the group consisting of Fragile X syndrome (FXS), Fragile X-associated tremor or ataxia (FXTAS), Fragile X-associated primary ovarian insufficiency (FXPOI), autism, mental retardation, cognitive impairment, and a modified X-chromosome. 
     
     
         3 . The method of  claim 1  wherein the epigenetic modification is methylation. 
     
     
         4 . The method of  claim 1  wherein a cell from the subject is a cultured or uncultured Chorionic Villi Sample (CVS) cell, a lymphoblast cell, a blood cell, buccal cell, epithelial cell, fibroblast cell, an amniocyte or an EBV transformed lymphoblast cell line. 
     
     
         5 . The method according to  claim 1  further comprising determining the length of (CGG)n expansion within the FMR genetic locus leading to a (CGG)n expansion pathology selected from the group consisting of a Gray Zone (GZ) pathology, a premutation (PM) pathology and a full mutation (FM) pathology. 
     
     
         6 . The method of  claim 1  wherein the trinucleotide expansion disorder is FXS or a related condition in a human subject, and wherein the screening is of an epigenetic modification in the FMR genetic locus at a location selected from the group consisting of (i) and (iii). 
     
     
         7 . A method for screening for an agent which modulates epigenetic modification of an FMR genetic locus in a mammalian cell including a human cell, said method comprising screening for a change relative to a healthy control in the extent of epigenetic change in (i) two or more of (a) an intron; (b) an intron/exon boundary; and/or (c) a splicing region; and/or (ii) approximately one seventh or greater of an intron including an intron/exon boundary and/or a splicing region of the FMR genetic locus; wherein the intron, intron/exon boundary and/or splicing region is selected from the group consisting of:
 (i) Fragile X-related Epigenetic Element 3 in FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:1 or a homolog thereof or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:1 or which hybridizes to SEQ ID NO:1 or its complementary form under medium stringency conditions;   (ii) intron 2 of FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:2 or a homolog thereof or a portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:2 or which hybridizes to SEQ ID NO:2 or its complementary form under medium stringency conditions; and   (iii) genomic FREE2 region as a whole or specific fragments of FREE2 including FREE2 (D), FREE2 (E) or FREES comprising the nucleotide sequence set forth in SEQ ID NO:48 or 49 or 47, respectively or a homolog or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:48 or 49 or 47 or which hybridizes to SEQ ID NO:48 or 49 or 47 or its complementary form under medium stringency conditions;   in the presence or absence of an agent to be tested wherein the agent is selected if it induces a change in extent of epigenetic modification.   
     
     
         8 . The method of  claim 7  wherein the epigenetic modification is methylation. 
     
     
         9 .- 15 . (canceled) 
     
     
         16 . The method according to  claim 1 , said method comprising extracting genomic DNA from a cell from said subject and subjecting the DNA to an amplication reaction using primers selective of (i) two or more of (a) an intron; (b) an intron/exon boundary; and/or (c) a splicing region; and/or (ii) approximately one seventh or greater of an intron including an intron/exon boundary and/or a splicing region within the genetic locus. 
     
     
         17 . The method of  claim 16  wherein the trinucleotide expansion disorder is FXS or a related condition selected from the group consisting of FXTAS, FXPOI, autism, mental retardation, a modified X-chromosome and cognitive impairment. 
     
     
         18 . (canceled) 
     
     
         19 . A kit for the use in a method of  claim 1  comprising primers which amplify a region with the FMR genetic locus, said region in:
 Fragile X-related Epigenetic Element 3 in FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:1 or a homolog thereof or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:1 or which hybridizes to SEQ ID NO:1 or its complementary form under medium stringency conditions; 
 (ii) intron 2 of FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:2 or a homolog thereof or a portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:2 or which hybridizes to SEQ ID NO:2 or its complementary form under medium stringency conditions; 
 (iii) genomic FREE2 region as a whole or specific FREE2 fragments including FREE2 (D), FREE2 (E) or FREES comprising the nucleotide sequence set forth in SEQ ID NO:48 or 49 or 47, respectively or a homolog or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:48 or 49 or 47 or which hybridizes to SEQ ID NO:48 or 49 or 47 or its complementary form under medium stringency conditions; 
 (iv) an intron, intron/exon boundary and/or splicing region downstream of intron 2 of FMR1 or a homolog thereof or a portion or fragment thereof; 
 (v) two or more of (a) an intron; (b) an intron/exon boundary; and/or (c) a splicing regions within the FMR genetic locus; and 
 (vi) approximately one seventh or greater of an intron including an intron/exon boundary and/or a splicing region within the FMR genetic locus. 
 
     
     
         20 . The kit of  claim 19  wherein the primers are selected from the group consisting of SEQ ID NOs:6 through 11. 
     
     
         21 . A computer program product for assessing progression of a pathological condition associated with the FMR locus in a subject, the product comprising:
 (1) assigning a value to one or more of:
 (a) change in of methylation or other epigenetic modification relative to a control in FREE3 of FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:1 or a homolog thereof or portion or part thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:1 or which hybridizes to SEQ ID NO:1 or its complementary form under medium stringency conditions; 
 (b) change of methylation or other epigenetic modification relative to a control in intron 2 of FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:2 or a homolog thereof or portion or part thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:2 or which hybridizes to SEQ ID NO:2 or its complementary form under medium stringency conditions; 
 (c) change in methylation or other epigenetic modification relative to a control in genomic FREE2 region as a whole or specific FREE2 fragments including FREE2 (D), FREE2 (E) or FREE3 comprising the nucleotide sequence set forth in SEQ ID NO:48 or 49 or 47, respectively or a homolog or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:48 or 49 or 47 or which hybridizes to SEQ ID NO:48 or 49 or 47 or its complementary form under medium stringency conditions; 
 (d) change of methylation in an intron, intron/exon boundary and/or splicing region downstream of intron 2 of FMR1 or a homolog thereof or a portion or fragment thereof; 
 (e) two or more of (i) an intron; (ii) an intron/exon boundary; (iii) a splicing region within the FMR genetic locus; 
 (f) approximately one seventh or greater of an intron including an intron/exon boundary and/or a splicing region within the FMR genetic locus; 
 (g) length of (CGG)n expansion within the FMR genetic locus when considered in combination with (a) and/or (b); 
 (h) general phenotype or clinical manifestations in subjects with a neurodevelopmental or neurodegenerative condition; 
 (i) behavioral assessment criteria associated with normal subjects, PM subjects, GZ subjects and FM subjects; 
 (j) cognitive ability; 
 (k) extent of transcription of genes within the FMR locus with the proviso that if any one of (d) through (k) is determined then one or more of (a) through (c) is also determined; 
   (2) means to converting the value to a code; and   (3) means to store the code in a computer readable medium and compare code to a knowledge database to determine whether the code corresponds to a pathological condition.   
     
     
         22 . A computer for assessing an association between extent of methylation or other epigenetic modification within the FMR locus, the FMR locus and progression of a disease condition wherein the computer is programmed by the program of  claim 21 . 
     
     
         23 . A method of identifying epigenetic profile in a population of human subjects indicative of a pathological condition associated with the FMR locus, said method comprising identifying a trinucleotide expansion disorder according to  claim 1  in a plurality of members of said population. 
     
     
         24 . A method of allowing a user to determine the status, prognosis and/or treatment response of a subject with respect to an FMR locus-associated pathology, the method including:
 (1) receiving data in the form of extent of methylation or other epigenetic modification at a site in:
 (i) Fragile X-related Epigenetic Element 3 in FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:1 or a homolog thereof or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:1 or which hybridizes to SEQ ID NO:1 or its complementary form under medium stringency conditions; 
 (ii) intron 2 of FMR1 comprising the nucleotide sequence set forth in SEQ ID NO:2 or a homolog thereof or a portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:2 or which hybridizes to SEQ ID NO:2 or its complementary form under medium stringency conditions; 
 (iii) genomic FREE2 region as a whole or specific FREE2 fragments including FREE2 (D), FREE2 (E) or FREES comprising the nucleotide sequence set forth in SEQ ID NO:48 or 49 or 47, respectively or a homolog or portion or fragment thereof defined by having at least 80% nucleotide sequence identity to SEQ ID NO:48 or 49 or 47 or which hybridizes to SEQ ID NO:48 or 49 or 47 or its complementary form under medium stringency conditions; 
 (iv) an intron, intron/exon boundary and/or splicing region downstream of intron 2 of FMR1 or a homolog thereof or a portion or fragment thereof; 
 (v) two or more of (a) an intron; (b) an intron/exon boundary; and/or (c) a splicing regions within the FMR genetic locus; and 
 (vi) approximately one seventh or greater of an intron including an intron/exon boundary and/or a splicing region within the FMR genetic locus; 
 wherein the extent of methylation or epigenetic modification provides a correlation to the presence, state, classification or progression of the pathology; 
   (2) transferring the data from the user via a communications network;   (3) processing the subject data via multivariate or univariate analysis to provide a disease index value;   (4) determining the status of the subject in accordance with the results of the disease index value in comparison with predetermined values; and   (5) transferring an indication of the status of the subject to the user via the communications network.