Treatment and diagnosis of epigenetic disorders and conditions

Abstract

A method identifies a trinucleotide expansion disorder in a mammalian subject. The method includes screening for a change relative to a healthy control in the extent of epigenetic modification within two or more of an intron, an intron/exon boundary, and/or a splicing region. 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 such a disorder. The intron, intron/exon boundary and/or splicing region can be Fragile X-related Epigenetic Element 3 in FMR1, intron 2 of FMR1, or genomic FREE2 region as a whole or specific fragments of FREE2 including FREE2 (D), FREE2 (E) or FREE3. A method detects methylation in Fragile X-related Epigenetic Element 2(D) (FREE2(D)) in genomic DNA of a human subject and a computer program assesses progression of a pathological condition associated with the FMR locus in a subject

What is claimed is: 
     
         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 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.   
     
     
         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 of  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 . A method of detecting methylation in Fragile X-related Epigenetic Element 2(D) (FREE2(D)) in genomic DNA of a human subject, the method comprising:
 (a) obtaining isolated genomic DNA from the human subject,   (b) amplifying wholly or partially FREE2(D) consisting of the nucleotide sequence of SEQ ID NO:48 or a modified SEQ ID NO:48 in which methylated cytosine(s) in SEQ ID NO:48 are converted to uracil(s) using a first primer complementary to a first region of SEQ ID NO:48 or to the modified SEQ ID NO:48 sequence in which methylated cytosine(s) have been converted to uracil(s) and a second primer complementary to a second region of SEQ ID NO:48 or to the modified SEQ ID NO:48 sequence in which methylated cytosine(s) have been converted to uracil(s), wherein the second primer is non-overlapping with the first primer; and   (c) detecting methylation in the wholly or partially amplified FREE2(D).   
     
     
         7 . The method of  claim 6  wherein the genomic DNA is isolated from a cell from the human subject selected from the group consisting of a cultured or uncultured Chorionic Villi Sample (CVS) cell, a lymphoblast cell, a blood cell, buccal cell, epithelial cell, fibroblast cell, an amniocyte and an EBV transformed lymphoblast cell line. 
     
     
         8 . The method of  claim 6 , wherein FREE2(D), consisting of the nucleotide sequence of SEQ ID NO: 48, is amplified using primers having the nucleotide sequences of SEQ ID NO: 43 and SEQ ID NO: 44. 
     
     
         9 . The method of  claim 6 , wherein the method further comprises determining the length of (CGG) n  expansion. 
     
     
         10 . The method of  claim 6 , wherein step (b) comprises amplifying at least a part of a modified SEQ ID NO:48 in which methylated cytosine(s) in SEQ ID NO:48 is/are converted to uracil(s), and wherein prior to step (b) the method comprises:
 deaminating cytosines in the isolated genomic DNA by treating the isolated genomic DNA with bisulfite for a time and under conditions sufficient to convert non-methylated cytosines to uracils,   
       wherein at least one of the first or second primers complementary to modified SEQ ID NO:48 in which methylated cytosine(s) in SEQ ID NO:48 are converted to uracil(s) comprises a dinucleotide or a trinucleotide selected from the group consisting of a TG, a CG and a CNG, wherein amplification using a primer comprising a TG indicates the presence of a non-methylated cytosine at the corresponding position in SEQ ID NO:48, and amplification using a primer comprising CG or CNG, where N indicates any nucleotide, indicates presence of a methylated cytosine at the corresponding position in SEQ ID NO:48. 
     
     
         11 . The method of  claim 6 , wherein prior to step (b), the method comprises digesting the genomic DNA with a methylation-sensitive restriction endonuclease, and the digested genomic DNA is amplified in step (b). 
     
     
         12 . A computer program product for assessing progression of a pathological condition associated with the FMR locus in a subject, the product comprising:
 (1) software configured to assign 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) software configured to convert the value to a code; and   (3) a computer readable medium to store the code and a knowledge database against which to compare the code,   wherein the computer program is further configured to determine whether the code corresponds to a pathological condition.