Methods and systems for detecting fragile X methylation

Abstract

A method and system for detecting methylation of the fragile X FMRI gene is disclosed. In certain embodiments, the DNA is fragmented, and then the assay uses methylation-specific immunoprecipitation to separate the genomic DNA into methylated and unmethylated fractions. In certain embodiments, both fractions are processed in parallel along with the original unfractionated DNA. In certain embodiments, the assay is performed on multiple samples using multi-well plates. After separation of the PCR products by capillary electrophoresis, a custom computerized calling tool can be used to quantitatively determine the methylation status.

Description

[Technical field]

[0001] (Field) Methods and systems for detecting methylation of the fragile X FMR1 gene are disclosed. [Background technology]

[0002] (background) Fragile X syndrome (FXS), formerly known as Martin-Bell syndrome, is the second most common cause of genetic psychiatric disability (after Down syndrome), affecting approximately 1 in 4000 males and 1 in 8000 females. "Fragile X" derives its name from a cytogenetic fragile site at Xq27.3 that appears when cells from affected individuals are cultured in folic acid-deficient medium. Affected males exhibit developmental delay and intellectual disability, mild dysmorphic features, macroorchidism, and high-pitched, comical speech. Females usually exhibit a less severe phenotype, which is typical of X-linked disorders. As expected for a neurodevelopmental disorder, IQ tends to decline with age. Many patients also exhibit subtle connective tissue abnormalities, attention-deficit hyperactivity disorder, and autistic-like behavior. The phenotype is associated with mutations in the FMR1 gene located at Xq27.3 (reviewed in Hagerman, R. et al., Fragile X syndrome, Nat Rev Dis Primers, 2017, 3:17065). FMR1 is a highly conserved gene consisting of 17 exons and spanning approximately 38 Kb. Within the 4.4 Kb FMR1 transcript, there is a CGG trinucleotide repeat region located in the 5' untranslated region (UTR). The gene encodes an RNA-binding protein that regulates protein translation of many genes involved in synaptic function in the nervous system. FXS is caused by FMR1 protein (FMRP) deficiency. For >98% of cases, loss of FMRP results from hypermethylation of the FMR1 5'UTR region, which is induced by a CGG repeat expansion (reviewed in Hagerman et al., 2017). However, challenges exist with current assay systems (e.g., Southern blot detection of FMR1 methylation status), due, for example, to processing time and the amount of input DNA required. Thus, there is a need for improved methods and systems for measuring methylation patterns associated with alterations in the FMR1 gene and such alterations that may result in FMRP deficiency. In particular, there is a need for methods and systems that can increase the accuracy and specificity of such assays. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Hagerman, R. et al., Fragile X syndrome, Nat Rev Dis Primers, 2017, 3:17065 Summary of the Invention [Means for solving the problem]

[0004] (Abstract) Methods and systems are disclosed for detecting methylation of the FMR1 gene of fragile X. The methods and systems can be embodied in a variety of ways.

[0005] In certain embodiments, a method is disclosed for determining methylation of the FMR1 gene in a sample from a subject, the method comprising the steps of: isolating total nucleic acid from the sample, the total nucleic acid including both methylated and unmethylated nucleic acid; contacting a first portion of the isolated total nucleic acid with a methyl-binding protein; isolating a portion of the unbound nucleic acid, thereby producing a nucleic acid fraction enriched for unmethylated nucleic acid; isolating the bound nucleic acid, thereby producing a nucleic acid fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acid; and determining the presence or absence of an FMR1 amplification product in both the fraction enriched for methylated nucleic acid and the fraction enriched for unmethylated nucleic acid.

[0006] Also disclosed are compositions, kits, and systems for carrying out the disclosed methods or any of the steps of the disclosed methods.

[0007] The present disclosure may be better understood with reference to the following non-limiting drawings. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 illustrates a method for determining the methylation status of fragile X (FMR1) according to an embodiment of the present disclosure.

[0009] [Diagram 2] FIG. 2 shows the results of sonication of DNA isolated from various sample types according to an embodiment of the present disclosure.

[0010] [Diagram 3]3 shows a typical sheared DNA size distribution for 24 fresh blood DNA samples according to an embodiment of the present disclosure. The x-axis shows size (bp) and the y-axis shows relative fluorescence units (rfu). The peaks labeled 1 and 20,000 are internal sizing standards that indicate the range of the analyzer.

[0011] [Figure 4] FIG. 4 is a format for binding methylated DNA to the methyl-CpG binding domain (MBD)-containing protein MBD2a according to an embodiment of the present disclosure.

[0012] [Diagram 5] FIG. 5 shows a workflow scheme for analysis of the FMR1 gene according to an embodiment of the present disclosure.

[0013] [Figure 6] FIG. 6 shows a method for determining the size of an FMR1 allele, either methylated, unmethylated or partial, according to an embodiment of the present disclosure.

[0014] [Figure 7] FIG. 7 illustrates a system for determining methylation status of fragile X (FMR1) according to an embodiment of the present disclosure.

[0015] [Figure 8] FIG. 8 illustrates various exemplary computer processing devices according to embodiments of the present disclosure.

[0016] [Figure 9]9 shows the assessment of the methylation status of an expanded 75 bp CGG repeat in a sample (AFC4) as a fully methylated allele according to an embodiment of the present disclosure. The y-axis shows rfu, and the sizes of the various CGG repeat (rpt) regions are shown. Total shows total DNA; Meth shows DNA enriched for methylated DNA, and Unmeth shows DNA enriched for unmethylated DNA.

[0017] [Figure 10] Figure 10 shows the assessment of the methylation status of an expanded 163 bp CGG repeat in a sample (BLC2) as a partially methylated allele (i.e., detected in both methylated and unmethylated DNA) according to an embodiment of the present disclosure. The y-axis shows rfu, and the average size of the different CGG repeat (rpt) regions is shown. Total shows total DNA; Meth shows DNA enriched for methylated DNA, and Unmeth shows DNA enriched for unmethylated DNA.

[0018] [Figure 11] 11 shows the assessment of the methylation status of the unstable expanded 162 bp CGG repeat allele and FM allele in a sample (BL7) as unmethylated according to an embodiment of the present disclosure. The y-axis shows rfu, and the average size of the different CGG repeat (rpt) regions is shown. Total shows total DNA; Meth shows DNA enriched for methylated DNA, and Unmeth shows DNA enriched for unmethylated DNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] (Detailed Description) Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to facilitate the description of the subject matter disclosed herein. Other definitions can be found throughout the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the subject matter described herein belongs.

[0020] (definition) Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements. Moreover, all ranges disclosed herein should be understood to include any and all subranges subsumed therein. For example, the recited range "1 to 10" should be considered to include any and all subranges between its minimum value of 1 and its maximum value of 10 (and both ends inclusive) (i.e., all subranges beginning with a minimum value of 1 or more (e.g., 1 to 6.1) and ending with a maximum value of 10 or less (e.g., 5.5 to 10)). Moreover, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0021] The terms "a," "an," and "the" as used in this application (including the claims) refer to "one or more." Thus, for example, reference to "a cell" includes a plurality of such cells unless the context clearly indicates to the contrary (e.g., a plurality of cells), and so forth.

[0022] The term "and / or", when used in a list of two or more items, means that any one of the listed items may be used alone or in combination with any one or more of the listed items. For example, the phrase "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The phrase "A, B and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0023] As used herein, the term "detectable moiety" or "detectable biomolecule" or "reporter" refers to a molecule that can be measured in a quantitative assay. For example, a detectable moiety can include an enzyme that can be used to convert a substrate into a product that can be measured (e.g., a visible product). Or, a detectable moiety can be a radioisotope that can be quantified. Or, a detectable moiety can be a fluorophore. Or, a detectable moiety can be a luminescent molecule. Or, other detectable molecules can be used.

[0024] The terms "labeled" and "labeled with a detectable agent or moiety" are used interchangeably herein to specify that an entity (e.g., a nucleic acid probe, an antibody) can be measured, for example, by detecting the label (e.g., visual detection of radioactivity, fluorescence, etc.) after binding to another entity (e.g., a nucleic acid, a polypeptide). The detectable agent or moiety can be selected so that it produces a signal that can be measured and whose intensity is related (e.g., proportional) to the amount of bound entity. A wide variety of systems for labeling and / or detecting nucleic acids are known in the art. Labeled nucleic acids can be prepared by incorporating or conjugating to a label that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. A label or labeling moiety may be directly detectable (i.e., it does not require any further reaction or manipulation in order to be detectable, e.g., a fluorophore is directly detectable), or it may be indirectly detectable (i.e., it is made detectable through reaction or binding with another detectable entity, e.g., a hapten is detectable by immunostaining after reaction with an appropriate antibody containing a reporter (e.g., a fluorophore). Suitable detectable agents include, but are not limited to, radionucleotides, fluorophores, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, aptamer beacons, and the like.

[0025] As used herein, the term "biological sample" or "sample" refers to a sample obtained from a biological source, including, but not limited to, an animal, cell culture, organ culture, etc. Suitable samples include whole blood, amniotic fluid, amniotic fluid cell culture, chorionic villus sampling, chorionic villus sample cell culture, saliva and buccal, as well as cell-free DNA, plasma, serum, urine, tears, cerebrospinal fluid, organ, hair, muscle, or other tissue samples. In certain embodiments, the sample contains prenatal nucleic acid.

[0026] As used herein, "subject" may include animals. Thus, in some embodiments, the biological sample is obtained from a mammal (including, but not limited to, a human or fetus, dog, cat, horse, rat, monkey, etc.). In some embodiments, the biological sample is obtained from a human subject. In some cases, the human subject is a pregnant woman. In some embodiments, the subject is prenatal (i.e., a fetal DNA sample obtained from a pregnant woman). In some embodiments, the subject is a patient, i.e., a living human presenting in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition.

[0027] As used herein, total nucleic acid is nucleic acid isolated from a sample that has not been subjected to selection for methylated nucleic acid sequences.

[0028] A nucleic acid fraction enriched for methylated nucleic acids is a nucleic acid sample that has been treated to enrich for methylated nucleic acid sequences relative to unmethylated nucleic acid sequences.

[0029] A nucleic acid fraction enriched for unmethylated nucleic acids is a nucleic acid sample that has been treated to enrich for nucleic acid sequences that are unmethylated relative to nucleic acid sequences that are methylated.

[0030] As used herein, PCR amplification of the FMR1 gene refers to polymerase chain reaction (PCR) amplification of at least a portion of the FMR1 gene using primers having sequences specific for at least a portion of the FMR1 gene or sequences specific for sequences upstream (i.e., 5') and / or downstream (i.e., 3') of the FMR1 gene to amplify the entire gene and additional upstream or downstream sequences contained in the primer sequences.

[0031] As used herein, a FRAX mePCR assay is a PCR assay used to determine whether FMR1 gene sequences are present in a nucleic acid fraction enriched for methylated nucleic acids or a nucleic acid fraction enriched for unmethylated nucleic acids, or both. In some embodiments, FRAX PCR uses GS-PCR.

[0032] As used herein, FRAX PCR assay refers to the PCR assay used to determine the size of FMR1 gene allele in subject.In some embodiments, FRAX PCR uses GS-PCR.The assay optionally includes determining the number of expanded CGG repeats and determining the gender of the subject from whom sample is obtained.

[0033] As used herein, GS-PCR refers to gene-specific (GS) PCR of the FMRI gene. GS-PCR may use primers located upstream (i.e., 5') and downstream (i.e., 3') of the FMR1 promoter region that contains the CGG repeats.

[0034] Expanded FMR1 allele refers to the allele of FMRI gene that has expanded CGG triplet repeat region compared to normal control.For example, expanded allele can be classified as premutation (PM), which is about 55 CGG repeats to 200 CGG repeats, and / or full mutation (FM), which is an allele of >200 CGG repeats.In general, the size of CGG repeat region that is < about 55 CGG repeats can be classified as normal.

[0035] (FMR1 methylation assay) Methods for detecting FMRI gene methylation have been disclosed. These methods can be embodied in a variety of ways.

[0036] In certain embodiments, a fragile X (FRAX) methylation PCR (mePCR) assay method and a system for performing the method are disclosed, which overcome many of the limitations of tests currently encountered in Southern blot (SB) analysis for methylation detection. In certain embodiments, the assay uses methylation-specific immunoprecipitation to separate genomic DNA into methylated and unmethylated fractions. In some embodiments, both fractions are processed in parallel along with the original unfractionated DNA. In certain embodiments, the assay is performed on multiple samples using multi-well plates. After separation of the PCR products by capillary electrophoresis or other size selection methods, a custom computerized calling method disclosed herein is used to quantitatively determine the methylation status. In addition to whole blood and prenatal samples, saliva and buccal samples have been validated to extend the utility of the FRAX mePCR assay.

[0037] Thus, in certain embodiments, a method is disclosed for determining methylation of the FMR1 gene in a sample from a subject, the method comprising the steps of isolating total nucleic acid from the sample, the total nucleic acid including both methylated and unmethylated nucleic acid; processing the nucleic acid to partially purify the unmethylated and methylated nucleic acids, thereby generating a nucleic acid fraction enriched for unmethylated nucleic acids and a nucleic acid fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acids; and determining the presence or absence of an FMR1 amplification product in both the fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids.

[0038] For example, a method for determining methylation of the FMR1 gene in a sample from a subject is disclosed, the method comprising the steps of isolating total nucleic acid from the sample, the total nucleic acid including both methylated and unmethylated nucleic acid; contacting a first aliquot of the isolated total nucleic acid with a methyl-binding protein; isolating a portion of the unbound nucleic acid, thereby producing a nucleic acid fraction enriched for unmethylated nucleic acid; isolating the bound nucleic acid, thereby producing a nucleic acid fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acid; and determining the presence or absence of an FMR1 amplification product in both the fraction enriched for methylated nucleic acid and the fraction enriched for unmethylated nucleic acid.

[0039] As described herein, the subject may be a human, for example, a pregnant woman. Or the subject may be a fetus carried by a pregnant woman. Suitable samples may include whole blood, amniotic fluid, amniotic fluid cell culture, chorionic villus sampling, chorionic villus sample cell culture, saliva and buccal, as well as cell-free DNA, plasma, serum, urine, tears, cerebrospinal fluid, organ, hair, muscle, or other tissue samples. In some embodiments, the sample comprises prenatal nucleic acid.

[0040] In certain embodiments, the method may further comprise determining the size of the FMR1 amplification product in at least one of the fractions enriched for the methylated nucleic acid and / or the fractions enriched for the unmethylated nucleic acid. In certain embodiments, the size of the amplification product is determined using primers located upstream (i.e., 5') and downstream (i.e., 3') of the FMR1 gene (i.e., GS-PCR). Thus, in certain embodiments, the size of the FMR1 gene (and promoter region) in either the methylated nucleic acid fraction or the unmethylated fraction may be determined. In certain embodiments, the method may further comprise determining the number of CGG repeats in the FMR1 gene for the amplification product in at least one of the fractions enriched for the methylated nucleic acid and the fractions enriched for the unmethylated nucleic acid. The number of CGG repeats may be determined based on the size of the GS-PCR product, for example, by comparing with the number of CGG repeats in a reference genome sequence, using capillary electrophoresis or an equivalent method. Thus, the disclosed methods may include determining whether the subject has either (i) partial methylation of FMR1; (ii) both methylated and unmethylated copies of FMR1; or (iii) full methylation of FMR1. Additionally and / or alternatively, the methods may include determining whether the subject has either (i) partial methylation of an expanded FMR1 allele; (ii) both a methylated and an unmethylated expanded FMR1 allele; or (iii) full methylation of an expanded FMR1 allele.

[0041] Also, in certain embodiments, the method may include comparing the size of the FMR1 gene in the methylated nucleic acid fraction or the unmethylated fraction with the size of the FMR1 gene detected in the total nucleic acid from the subject. In such embodiments, the method may include PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the total nucleic acid, and determining the size of at least one FMR1 amplification product in the total nucleic acid. In certain embodiments, the method may include determining the number of CGG repeats in the FMR1 gene for at least one amplification product obtained from the total nucleic acid fraction. In certain embodiments, determining the size of the FMR1 amplification product in at least one of the fractions enriched for the methylated nucleic acid and / or the unmethylated nucleic acid comprises comparing the size of the amplification product for the fraction enriched for the methylated nucleic acid and the fraction enriched for the unmethylated nucleic acid with the size of at least one FMR1 amplification product for the total nucleic acid. Additionally and / or alternatively, the method may include assessing that the size of the FMR1 amplification product in the fraction enriched for the methylated nucleic acid and the fraction enriched for the unmethylated nucleic acid is the size of at least one FMR1 amplification product for the total nucleic acid.

[0042] In certain embodiments, at least one primer used to generate the FMR1 amplification product is labeled with a detectable moiety. For example, in certain embodiments, at least one primer used to generate an amplification product from the FMR1 gene from the methylation enriched fraction and / or the non-methylation enriched fraction and / or the total nucleic acid fraction has the same sequence but is labeled with a different detectable moiety.

[0043] Any of a wide variety of detectable agents can be used in the practice of the present disclosure. Suitable detectable agents include, but are not limited to, various ligands, radionucleotides; fluorescent dyes; chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (e.g., dyes, colloidal gold, etc.); biotin; digoxigenin; haptens; and proteins for which antisera or monoclonal antibodies are available. Below are some non-limiting examples of some detectable moieties that can be used.

[0044] (Fluorescent dye) In certain embodiments, the detectable moiety is a fluorescent dye. Many known fluorescent dyes of a wide variety of chemical structures and physical characteristics are suitable for use in the implementation of the present disclosure. The fluorescent detectable moiety can be stimulated by a laser, with the emitted light being captured by a detector. The detector can be a charge-coupled device (CCD) or a confocal microscope, which records its intensity.

[0045] Suitable fluorescent dyes include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), hexachloro-fluorescein (HEX), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Q-DOTS, Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.), Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes (e.g., CY-3, CY-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, etc.), IRDye (e.g., IRD40, IRD 700, IRD 800, etc.), and the like.For more detailed examples of suitable fluorescent dyes and methods for attaching fluorescent dyes to other chemical entities (e.g., proteins and peptides), see, e.g., "The Handbook of Fluorescent Probes and Research Products," 9th Edition, Molecular Probes, Inc., Eugene, OR. Preferred properties of fluorescent labeling agents include high molar absorption coefficients, high fluorescence quantum yields, and photostability. In some embodiments, the labeling fluorophores exhibit absorption and emission wavelengths in the visible light (i.e., between 400 nm and 750 nm) range, but not in the ultraviolet (i.e., below 400 nm) range of the spectrum.

[0046] The detectable moiety may comprise more than one chemical entity, such as in fluorescence resonance energy transfer (FRET). Resonance transfer results in an overall enhancement of its emission intensity. See, for example, Ju et al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:4347. To achieve resonance energy transfer, a first fluorescent molecule (the "donor" fluor) absorbs light and transfers it to a second fluorescent molecule (the "acceptor" fluor) through the resonance of excited electrons. In one approach, both donor and acceptor dyes can be linked together and attached to an oligo primer. Methods for linking donor and acceptor dyes to nucleic acids are described, for example, in U.S. Pat. No. 5,945,526. Dye donor / acceptor pairs that can be used include, for example, fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, and fluorescein / QSY 7 dyes. Many of these dyes are also commercially available, for example, from Molecular Probes Inc. (Eugene, Oreg.). Suitable donor fluorophores include 6-carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and the like.

[0047] (radioactive isotope) In certain embodiments, the detectable moiety is a radioisotope. For example, the molecule can be radiolabeled (i.e., contain one or more atoms replaced by an atom having an atomic mass or mass number different from that normally found in nature), or an isotope can be bound to the molecule. Non-limiting examples of isotopes that may be incorporated into the molecule include hydrogen isotopes, carbon isotopes, fluorine isotopes, phosphorous isotopes, copper isotopes, gallium isotopes, yttrium isotopes, technetium isotopes, indium isotopes, iodine isotopes, rhenium isotopes, thallium isotopes, bismuth isotopes, astatine isotopes, samarium isotopes, and lutetium isotopes (i.e., 3H, 13C, 14C, 18F, 19F, 32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, 111In, 125I, 123I, 129I, 131I, 135I, 186Re, 187Re, 201T1, 212Bi, 213Bi, 211At, 153Sm, 177Lu).

[0048] (Dendrimers) In some embodiments, signal amplification is achieved using labeled dendrimers as detectable moieties (see, e.g., Physiol Genomics, 3:93-99, 2000). Fluorescently labeled dendrimers are available from Genisphere (Montvale, NJ). These can be chemically conjugated to oligonucleotide primers by methods known in the art.

[0049] In some embodiments, the detectable moiety is a dye, such as a fluorescent dye. In certain embodiments, the primers used to amplify the FMR1 gene in methylated nucleic acid have the same sequence, but one of the primers (e.g., the reverse primer) can be labeled with a different dye. Thus, in certain embodiments, the fluorescent dye used to detect the amplification product from the fraction enriched for unmethylated nucleic acid and / or the total nucleic acid is 6-carboxyfluorescein (FAM), and the dye used to detect the amplification product from the fraction enriched for methylated nucleic acid is hexachlorofluorescein (HEX).

[0050] Thus, in certain embodiments, the primers used to amplify the FMR1 gene in unmethylated nucleic acid include a forward primer FRAX-F1 (SEQ ID NO: 1) having the nucleic acid sequence 5'-GCT CAG CTC CGT TTC GGT TTC ACT TCC GGT-3' and a reverse primer having the sequence of SEQ ID NO: 4 (5'-AGC CCC GCA CTT CCA CCA CCA GCT CCT CCA-3'). In certain embodiments, the reverse primer is labeled at its 5' end with the dye FAM or the dye HEX, or a different detectable moiety. Additionally or alternatively, the forward primer (SEQ ID NO: 1) can be labeled with FAM or HEX (or a different detectable moiety).

[0051] Thus, in an embodiment, the reverse primers used to amplify the FMR1 gene in unmethylated nucleic acids are forward primers FRAX-F1 (SEQ ID NO: 1) and FRAX-R-6FAM (SEQ ID NO: 2) 5'-FAM-AGC CCC GCA CTT CCA CCA CCA GCT CCT CCA-3' (Table 1). Also, in a particular embodiment, the primers used to amplify the FMR1 gene in methylated nucleic acids include forward primer FRAX-F1 (SEQ ID NO: 1), but its reverse primer is labeled with HEX. Thus, in an embodiment, the reverse primer used to amplify the FMR1 gene in methylated nucleic acids is FRAX-R-HEX (SEQ ID NO: 3) 5'-HEX-AGC CCC GCA CTT CCA CCA CCA GCT CCT CCA-3' (Table 2). Alternatively, other combinations of primers of SEQ ID NO: 1 and primers of SEQ ID NO: 4 labeled with different moieties may be selected. [Table 1] [Table 2]

[0052] In certain embodiments, to optimize binding of the nucleic acid to the methyl-binding protein, the nucleic acid is fragmented into fragments having a size ranging from about 2.4 kilobase pairs (kb) to 3.2 kilobase pairs (kb) or about 2.5 kb or about 3 kb in size prior to contacting the nucleic acid with the methyl-binding protein. Or fragments of other sizes can be used as disclosed herein. Embodiments for generating nucleic acid fragments are disclosed in more detail herein.

[0053] In certain embodiments, the method is performed after an initial assessment of whether the sample contains at least one FMR1 allele that appears to exhibit an extension of the 5' upstream region, for example, by an extension of a CGG trinucleotide repeat region located in the 5' untranslated region (UTR) of the gene. Thus, the method may include a step of performing a PCR assay to determine the number and / or location of CGG repeats in the FMR1 gene in nucleic acid from the sample. For example, in certain embodiments, the method includes a step of PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the total nucleic acid fraction, and determining the size of the FMR1 amplification product (FRAX PCR). If additional assessment appears to be warranted (e.g., the sample contains PM or FM), the sample may be further assayed by FRAX me-PCR.

[0054] In some embodiments, the total nucleic acid can be fragmented for use in FRAX me-PCR assay. For example, in certain embodiments, the total nucleic acid can be fragmented by sonication, enzyme digestion, or other similar methods used to fragment nucleic acids. The method can further include a step of confirming the number of CGG repeats in the FMR1 gene for the amplification products obtained from the total nucleic acid fraction. In certain embodiments, the method can include a step of determining the size and amount of each allele peak using GS-PCR (i.e., GS-PCR). The method can also include a step of calculating the average number of CGG repeats for each allele from the GS-PCR data, and then using the calculated peak sizes as peaks of interest measured by FRAX me-PCR, i.e., determining whether the extended allele is methylated or unmethylated. In certain embodiments, the analysis does not include peaks from FRAX me-PCR that are not found in FRAX PCR from total nucleic acid. As described herein, in certain embodiments, determining the approximate size of the FMR1 gene may include amplification (e.g., GS-PCR) using primers located 5' and 3' of the FMRI gene and upstream region. In one embodiment, a primer having the sequence of SEQ ID NO: 1 for the forward primer and a primer having the sequence of SEQ ID NO: 2 for the reverse primer are used for GS-PCR (i.e., FRAX PCR) from total DNA.

[0055] Also, in certain embodiments, mosaicism in the sample is identified using FRAX PCR.For example, mosaicism can be determined by identifying the number of characteristic peaks from FRAX PCR.Since each peak represents one allele, it is expected to detect one allele in male samples or two alleles in female samples, respectively.Mosaicism can be identified when more than one allele is detected in male samples or more than two alleles are detected in female samples, respectively.

[0056] In certain embodiments, the intended clinical use of the FRAX mePCR assay is to determine the methylation status of premutation (PM), which is an allele with approximately 55 CGG repeats to 200 CGG repeats, and / or full mutation (FM), which is an allele with >200 CGG repeats. This may be important because methylation status may affect clinical severity for both PM carriers as well as FM carriers. Thus, while unmethylated FM alleles may still produce FMR1 protein, it is rare to find an FM allele carrier with an unmethylated status. In certain embodiments, the method is directed to Fragile X carrier screening and diagnostic testing, including at-risk prenatal specimens.

[0057] Thus, in certain embodiments, prior to determining the methylation status at the FMR1 locus, PCR amplification is used to determine the extent of CGG expansion and the sex of the individual whose FMR1 status is being evaluated. Thus, in some embodiments, prior to testing in the FRAX mePCR assay, a determination of whether the subject may contain either no mutation (i.e., no expansion, premutation i.e., 55 CGG repeats to 200 CGG repeats) or full mutation (i.e., >200 CGG repeats) and a determination of the sex are performed. The determination of the nature of the CGG expansion and / or the sex may, in certain embodiments, include three parts: i) gene-specific PCR (GS-PCR) to determine the total number of repeats in each allele; (ii) triplet-primed PCR (TRP-PCR) to screen for repeat expansions, if necessary; and (iii) differential detection PCR. In certain embodiments, the initial GS-PCR may use labeled primers to facilitate analysis (e.g., using equipment used for Sanger sequencing). Alternatively, unlabeled primers can be used for detection using, for example, a fragment analyzer, a bioanalyzer, etc. If the sample is determined to have either a premutation (PM) or full mutation (FM), an assessment of the methylation status for each FMR1 allele can be performed.

[0058] In certain embodiments, the methyl-binding protein is a bifunctional polypeptide comprising (i) an Fc portion of an antibody, (ii) a short flexible peptide linker, and (iii) a DNA-binding domain of MBD2 protein. In certain embodiments, the methyl-binding protein is provided by New England Biolabs (Ipswich, MA) as the EpiMark® Methylated DNA Enrichment Kit. The EpiMark Kit provides the methyl-CpG-binding domain of human MBD2 protein fused to the Fc tail of human IgG1 (MBD2-Fc). Alternatively, other methyl-binding proteins (e.g., Active Motif's MethylCollector™ Ultra available from Active Motif, Inc. (Carlsbad, CA)) may be used. Alternatively, other methyl-binding proteins may be used.

[0059] In certain embodiments, the amplification products can be analyzed by capillary electrophoresis. Or other size fractionation techniques can be used, such as gel electrophoresis or melting curve analysis (i.e., the size of PCR products can be determined by their melting temperature). The results can be used to determine whether the subject has no methylation, partial methylation of FMR1 (having both methylated and unmethylated copies of FMR1), or full methylation (both alleles are methylated), for each allele identified in the total nucleic acid. The method can further include determining the size of the amplification products of the enriched methylated or enriched unmethylated nucleic acids.

[0060] 1 shows a schematic diagram of an embodiment of an assay method 100. The assay may thus include a step 102 of providing a sample (e.g., a maternal and / or prenatal sample) from a subject to be screened for alleles that may be indicative of risk of fragile X.

[0061] The sample may be processed to isolate nucleic acids (104). In one embodiment, the nucleic acid is DNA. In certain embodiments, the DNA may be cell-free DNA, for example, from prenatal testing. Samples that may be used include, but are not limited to, whole blood, plasma, serum, amniotic fluid, amniotic fluid (AF) cell culture, chorionic villus sampling (CVS), chorionic villus sample cell culture, saliva samples, and buccal samples.

[0062] In certain embodiments, the nucleic acid is treated to generate fragments of a particular size or within a particular size range that efficiently binds to methyl-binding proteins (106). In certain embodiments, the nucleic acid is sonicated to generate fragments that are about 2.5 kb to 3 kb in size. Alternatively, nucleic acid fragments can be generated by other methods (e.g., digestion with restriction enzymes). For example, conditions can be used such that the nucleic acid is fragmented to a size range of 1 kb to 5 kb, or 2 kb to 4 kb, or about 2.2 kb to 3.5 kb, or about 2.4 kb to 3.2 kb, or about 2.5 kb or 3 kb. In certain embodiments, the optimal size is about 2.5 kb. In other embodiments, the optimal size is about 3.0 kb. Thus, the optimal fragment size can vary based on the size of the PCR product expected based on the largest FMR1 allele found for the sample. Thus, in certain embodiments, generating fragments of a particular size can increase the specificity of the assay. For example, Figures 2 and 3 show examples of nucleic acid (DNA) generated using a COVARIS® 3kb multi-well sonication plate on a COVARIS® R230 sonicator. Thus, as shown in Figure 2, DNA from fresh blood, 4-5 week old blood, prenatal CVS, prenatal AF, CVS cultured cells, AF cultured cells, saliva and buccal swabs can be reproducibly sonicated into fragments with an average size of about 3kb. In certain embodiments, shearing is highly efficient, with >90% of the DNA being sheared for all samples. Figure 3 shows a typical analysis of the size distribution of sheared DNA analyzed by an Agilent fragment analyzer using the High sensitivity Large Fragment Analysis Kit DNF-464. The electropherogram shows the results of 24 fresh blood DNA samples.

[0063] Referring back to FIG. 1, after preparing nucleic acid fragments of appropriate size, the nucleic acid can be incubated with a methyl-binding protein to separate methylated from unmethylated nucleic acids (108). In certain embodiments, the methyl-binding protein is cross-linked to a solid support. For example, the methyl-binding protein can be cross-linked to hydrophilic magnetic beads. In certain embodiments, the methyl-binding protein is provided by New England Biolabs (Ipswich, MA) as the EpiMark® Methylated DNA Enrichment Kit and used according to the manufacturer's instructions. Alternatively, other methods of enriching methylated and unmethylated nucleic acids can be used.

[0064] In certain embodiments, the supernatant enriched for unmethylated nucleic acids may then be collected prior to washing the complexed methylated nucleic acid:methyl-binding protein (110). After washing the MBP:methylated nucleic acid complex, the nucleic acid fraction enriched for methylated nucleic acids is eluted from the MBP (112). In certain embodiments, the methylated DNA is eluted in 30 μL of 10 mM Tris-HCL (pH 8.0), which may be stored at −20° C. for up to about 1 week.

[0065] In certain embodiments, PCR amplification is used to determine the extent of CGG extension using a second aliquot of the total unsheared DNA (114). This may be done to confirm the results from the initial screening of the sample. Or in certain embodiments, an aliquot of the total sheared DNA (unfractionated for either methylated or unmethylated) may be used. This may be done in parallel with meFRAX PCR (116). Thus, as discussed in more detail herein, amplification of the unmethylated FMR1 locus may use primers FRAX-F1 and FRAX-R-6FAM (i.e., SEQ ID NO:1 and SEQ ID NO:2, respectively). Amplification of the methylated FMR1 locus may use primers FRAX-F1 and FRAX-R-6HEX (i.e., SEQ ID NO:1 and SEQ ID NO:3, respectively). Amplification of the total (unfractionated DNA) may use the same primers used for the unmethylated DNA, i.e., FRAX-F1 and primers FRAX-R-6FAM (i.e., SEQ ID NO:1 and SEQ ID NO:2, respectively).

[0066] The FRAX mePCR assay can then be used (a) to assess the methylation status of each FMRI allele detected in the sample, and, if necessary, (b) to confirm the initial GS-PCR results from the FRAX PCR assay (116). Those results can then be provided to the subject or to the subject's healthcare provider (118).

[0067] Thus, in certain embodiments, the FRAX mePCR assay begins with enrichment of unmethylated and methylated DNA from total genomic DNA fragmented by sonication. As shown in FIG. 4, the enrichment process may involve specific methylated DNA binding by the methyl-CpG-binding domain (MBD)-containing protein MBD2a (Gebhard et al., Rapid and sensitive detection of CpG-methylation using methyl-binding (MB)-PCR, Nucleic Acids Res., 2006;34:e82; Schilling et al., 2007, Comparative analysis of tissue-specific promoter CpG methylation, Genomics, 2007;90:314-23). ​​In certain embodiments, the methyl-binding protein is provided by New England Biolabs (Ipswich, MA) as the EpiMark® Methylated DNA Enrichment Kit. In certain embodiments, the prepared MBD-bound magnetic beads may be stored at 4° C. for about one week.

[0068] Figure 5 shows another embodiment of the workflow for the method of the present disclosure. In a particular embodiment, both methylated and unmethylated DNA fractions from the same sample are analyzed by gene-specific PCR (GS-PCR) and capillary electrophoresis (CE). To ensure that the two fractions are accurately distinguished and visualized during analysis, the methylated and unmethylated PCR products are end-labeled with different fluorophores (i.e., HEX and FAM, respectively). The capillary electrophoresis (CE) conditions used for the FRAX mePCR assay can be the same as those in the FRAX PCR assay for both short (GS-S) and long (GS-L) injections to separate CGG repeat sizes ranging from normal length to full mutation (>200 CGGs). In certain embodiments, the peak calling tool (i.e., software, such as but not limited to GeneMapper software) used to detect peaks from CE for GS-PCR in the FRAX PCR assay is also used in the FRAX mePCR assay to detect the size of all FMR1 alleles found in the unfractionated total DNA sample. The methylation status of each FMR1 allele is then evaluated based on whether the allele detected in the total DNA is also identified in the methylated and unmethylated DNA fractions.

[0069] Still referring to FIG. 5, in certain embodiments, a portion of the nucleic acid (e.g., 1 μg of DNA) is sheared (e.g., sonicated) to generate fragments of a certain size or within a certain size range that efficiently bind to methyl-binding proteins, as described above. In certain embodiments, 75 μL of the 200 μL fragmented DNA can be used, and the remainder can be stored at −20° C. for at least one week. In certain embodiments, fragments of about 3 kb are used. Both fractions (methylated and unmethylated) are then analyzed by gene-specific PCR (GS-PCR) (also called me-FRAX PCR). To ensure that the two fractions are accurately distinguished and visualized during analysis, the methylated and unmethylated PCR products are end-labeled with different fluorophores (i.e., HEX and FAM, respectively). Thus, the GS-PCR primers for unmethylated PCR can be SEQ ID NO:1 and SEQ ID NO:2, and the GS-PCR primers for methylated PCR can be SEQ ID NO:1 and SEQ ID NO:3. As described herein, a second aliquot of total DNA (i.e., unsheared and unfractionated into methylated or unmethylated) can be amplified with primers of SEQ ID NO:1 and SEQ ID NO:2 to determine the size of the repeat region (i.e., FRAX PCR). In this way, the results of the "overall" GS-PCR should be consistent with the initial screen using FRAX PCR.

[0070] In certain embodiments, after data collection (e.g., using 3730xl Data Collection v.3.0 software or other analytical data processing software), the CE data for GS-PCR is analyzed using GeneMapper v.4.0 software (ABI / Thermo Fisher) or other analytical data processing software. One embodiment of an analytical method 600 that can be used is shown in FIG. 6. Thus, in certain embodiments, the software outputs the analyzed size of each PCR amplicon as peak size and the amount as peak height, respectively, which are then processed by the FRAX mePCR methylation calling tool (602). Thus, in certain embodiments, the mePCR algorithm first checks the sizing standard and peak height to ensure that the GS-PCR CE data quality for FRAX PCR with total DNA and FRAX mePCR is acceptable for analysis (604). The method can then use the GS-PCR peak data of samples derived from total DNA to calculate the number of CGG repeats for each allele (606). Using these CGG repeat numbers, the algorithm may then scan the GS-PCR CE data of the methylated DNA fraction in the HEX channel and the GS-PCR CE data of the unmethylated DNA fraction in the FAM channel, respectively, to identify corresponding peaks that are higher than the allele-specific peak height threshold (608). In one embodiment, the methylation status calling algorithm does not identify additional peaks in either the methylated or unmethylated fractions beyond those detected using total DNA (608). In this manner, the algorithm functions similarly to the process currently used with Southern blot (SB) analysis, where a CGG allele detected by GS-PCR in a FRAX PCR assay is confirmed and its methylation status is determined. Detection of the same peak in total DNA and in either the methylated or unmethylated fraction, but not both, indicates a fully methylated or unmethylated status for the allele, respectively.If the same peak is present in both the methylated and unmethylated fractions as well as in the total DNA, the allele is called as partially methylated (612). At this point, the results can be reported (614).

[0071] As discussed herein, each of these analysis steps may be controlled by a computer or data processor 800 using a non-transitory computer-readable recording medium containing instructions which, when executed on the data processor, cause the data processor to perform any one of these steps.

[0072] Compositions and Kits Also disclosed herein are compositions and kits for carrying out any of the disclosed methods or for implementing any of the components and / or stations of the disclosed systems.In some embodiments, the compositions may include an oligonucleotide having a sequence as shown in SEQ ID NO:1 or SEQ ID NO:4.In some embodiments, the oligonucleotide having a sequence as shown in SEQ ID NO:1 or SEQ ID NO:4 may be labeled with a detectable moiety.For example, in certain embodiments, the compositions may include at least one primer having a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0073] Also disclosed are kits for carrying out any of the above or below described method embodiments or for using any of the above or below described composition embodiments. In certain embodiments, the kits may include an oligonucleotide having a sequence as set forth in SEQ ID NO:1 or SEQ ID NO:4. In certain embodiments, the oligonucleotide having a sequence as set forth in SEQ ID NO:1 or SEQ ID NO:4 may be labeled with a detectable moiety. For example, in certain embodiments, the kits may include at least one primer having a sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. The kits may further include nucleic acid molecules providing positive and / or negative controls for assaying the methylation status of the FMRI gene and / or the FMR1 gene. Thus, the kits may include a nucleic acid having an extended FMR1 repeat region (i.e., <55 CGG repeats) as a negative control. Additionally and / or alternatively, the kits may include a nucleic acid having an extended FMR1 repeat region (e.g., a premutation (PM) that is between about 55 CGG repeats and 200 CGG repeats, and / or a full mutation (FM) that is an allele of >200 CGG repeats) as a positive control. The negative and / or positive controls may be pre-characterized as being fully methylated, partially methylated, or unmethylated. The kit may further comprise a reagent for enrichment of methylated and / or unmethylated nucleic acids. For example, the kit may comprise a methyl-binding protein. The methyl-binding protein may comprise (i) an Fc portion of an antibody; (ii) a short flexible peptide linker; and (iii) a DNA-binding domain of MBD2 protein. The kit may further comprise instructions for use.

[0074] (System for FMR1 methylation assay) Also disclosed is a system for detecting methylation of the FMR1 gene of fragile X. In certain embodiments, the system can perform any one of the steps of the disclosed method. The system can be embodied in various ways. Furthermore, each of the stations and / or components described herein can be a separate station or component, or can be coordinated and / or controlled by a different station or component.

[0075] For example, in certain embodiments, the system may include a station or component for isolating total nucleic acid from the sample. The system may also include a station or component for fragmenting the nucleic acid to a particular size to optimize binding of methylated nucleic acid to MBP. For example, conditions may be used such that the nucleic acid is fragmented to a size range of 1 kb to 5 kb, or 2 kb to 4 kb, or about 2.5 kb to 3.5 kb. The system may include a component or station for enriching methylated and / or unmethylated nucleic acid. For example, the system may further include a component or station for contacting a first portion of the isolated total nucleic acid with a methyl-binding protein. The system may further include a component or station for collecting a fraction of nucleic acid enriched for methylated and / or unmethylated nucleic acid. The system may further include a station or component for performing PCR amplification of the FMR1 gene. In one embodiment, the amplification is FRAX mePCR to determine whether FMR1 gene sequences are present in a nucleic acid fraction enriched for methylated nucleic acids and / or a nucleic acid fraction enriched for unmethylated nucleic acids, or both.

[0076] The system may further comprise a component or station for determining whether the extended allele detected in the total nucleic acid from the subject is methylated or unmethylated. Thus, in certain embodiments, the system comprises software (and / or data processor) that outputs the analyzed size of each PCR amplicon from FRAX PCR and / or meFRAX PCR as peak size and the amount as peak height, respectively, which are then processed by FRAX mePCR methylation calling software tool. In certain embodiments, the mePCR algorithm first checks the size standard and peak height to ensure that the CE data quality of the FRAX PCR and / or meFRAX PCR is acceptable for analysis. Then, in certain embodiments, the method may calculate the number of CGG repeats for each allele using the GS-PCR peak data of the sample from the total DNA. Using these CGG repeat numbers, the algorithm can then scan the GS-PCR CE data of the methylated DNA fraction in the HEX channel and the GS-PCR CE data of the unmethylated DNA fraction in the FAM channel, respectively, to identify corresponding peaks that are higher than the allele-specific peak height threshold. In an embodiment, the methylation status software does not identify additional peaks in either the methylated or unmethylated fractions, other than the peaks detected using total DNA. In this way, the CGG alleles detected by GS-PCR in the FRAX PCR assay from total nucleic acids are confirmed and their methylation status is determined. Detection of the same peak in total DNA and in either the methylated or unmethylated fraction, but not both, indicates a fully methylated or unmethylated status for the allele, respectively. If the same peak is present in both the methylated and unmethylated fractions and in total DNA, the allele is called as partially methylated. The system can further include a device for reporting the results to the subject and / or the subject's health care provider.

[0077] Reagents used to perform any of the steps of the method are also included as part of the disclosed system. For example, in certain embodiments, the reagents include at least one primer having the sequence of SEQ ID NO: 1 to SEQ ID NO: 3 disclosed herein.

[0078] For example, as shown in FIG. 7, a system 700 may include a station or component 702 for obtaining or processing a sample for assessment of FMR1 extension and / or methylation in a sample from a subject.

[0079] The system may further include a station or component 704 for isolating total nucleic acid from the sample. The system may also include a station or component for fragmenting the nucleic acid to a particular size. In certain embodiments, the nucleic acid may be fragmented to a size that optimizes binding of methylated nucleic acid to MBP (706). In certain embodiments, the nucleic acid is sonicated to generate fragments that are about 3 kb in size. For example, conditions may be used such that the nucleic acid is fragmented to a size range of 1 kb to 5 kb, or 2 kb to 4 kb, or about 2.2 kb to 3.5 kb, or about 2.4 kb to 3.2 kb, or about kb or about 3 kb.

[0080] The system may further include a component or station for enriching nucleic acids for methylated and / or unmethylated nucleic acids and collecting a fraction of nucleic acids enriched for methylated and / or unmethylated nucleic acids. For example, after preparing nucleic acid fragments of appropriate size, the nucleic acids may be incubated with a methyl-binding protein to separate methylated from unmethylated nucleic acids (708). In certain embodiments, the methyl-binding protein is cross-linked to a solid support. For example, the methyl-binding protein may be cross-linked to hydrophilic magnetic beads. Then, in certain embodiments, the supernatant containing the mostly unmethylated nucleic acids may be collected before washing the complexed methylated nucleic acid:methyl-binding protein to isolate the methylated nucleic acids as disclosed herein. Thus, the system may include a station or component 710 for collecting a fraction enriched for methylated and / or unmethylated nucleic acids.

[0081] The system may further include a station or component 712 for performing PCR amplification of the FMR1 gene. In an embodiment, the amplification is FRAX mePCR to determine whether FMR1 gene sequences are present in a nucleic acid fraction enriched for methylated nucleic acids and / or a nucleic acid fraction enriched for unmethylated nucleic acids, or both. Additionally, the station or component may include FRAX PCR to determine the size of the FMR1 gene allele in a subject, where the assay optionally includes determining the number of expanded CGG repeats and the gender of the subject from whom the sample was obtained.

[0082] In certain embodiments, PCR amplification products from FRAX mePCR and / or FRAX PCR are analyzed by capillary electrophoresis (CE) or other size separation step, and thus the system may include a component or station 714 for CE.

[0083] Analysis of the data may be performed using an automated analysis system, such as those disclosed herein, and thus the system may include a station or component 716 for data analysis.

[0084] As shown in FIG. 7, any of the stations and / or components of the system may be automated, robotically controlled, and / or at least partially controlled by a computer (e.g., data processor) 800 and / or programmable software. For example, the stations and / or components for FRAX mePCR or FRAX PCR, or CE, or data analysis may be controlled by a computer. Thus, the system may include a computer program product tangibly embodied in a non-transitory machine-readable recording medium, the computer program product including instructions configured to execute the system or any portion (e.g., station or component) of the system and / or to perform a step or steps of the method of any of the disclosed embodiments. In some embodiments, a system is provided that includes one or more data processors and a non-transitory computer-readable recording medium including instructions that, when executed on the one or more data processors, cause the one or more data processors to perform some or all of one or more methods or processes disclosed herein and / or to perform any portion of the system disclosed herein.

[0085] For example, a system is disclosed that includes one or more data processors and a non-transitory computer readable storage medium including instructions that, when executed on the one or more data processors, cause the one or more data processors to perform operations that direct at least one of the following steps of determining methylation of the FMR1 gene in a sample from a subject: isolating total nucleic acid from the sample, the total nucleic acid including both methylated and unmethylated nucleic acid; contacting a first aliquot of the isolated total nucleic acid with a methyl-binding protein; isolating a portion of the nucleic acids that are not bound to the methylated nucleic acids, thereby producing a nucleic acid fraction enriched for unmethylated nucleic acids; isolating the bound nucleic acids, thereby producing a nucleic acid fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acids; and determining the presence or absence of an FMR1 amplification product in both the fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids.

[0086] The data processor and non-transitory computer readable medium may further include instructions for analyzing data from FRAX me-PCR. Thus, the data processor may include a non-transitory computer readable medium including instructions for performing the steps of providing the size and amount of each PCR amplicon from FRAX PCR and / or meFRAX PCR as peak size and peak height. In certain embodiments, the analysis checks the size standard and peak height to ensure that the GS-PCR CE data quality is acceptable for analysis. The method may then use the GS-PCR peak data of the sample from FRAX PCR and / or meFRAX PCR to calculate the number of CGG repeats for each allele. Using these CGG repeat numbers, the method may then scan the GS-PCR CE data of the methylated DNA fraction in the HEX channel and the GS-PCR CE data of the unmethylated DNA fraction in the FAM channel, respectively, to identify corresponding peaks that are higher than the allele-specific peak height threshold. In one embodiment, the methylation status calling algorithm does not identify additional peaks in either the methylated or unmethylated fractions, other than the peaks detected using total DNA. In this way, the CGG allele detected by GS-PCR in the FRAX PCR assay is confirmed and its methylation status is determined. Detection of the same peak in total DNA and in either the methylated or unmethylated fraction, but not both, indicates a fully methylated or unmethylated status for the allele, respectively. If the same peak is present in both the methylated and unmethylated fractions and in total DNA, the allele is called as partially methylated. The system may further include instructions to report the results.

[0087] Also disclosed is a computer program product tangibly embodied in a non-transitory machine-readable recording medium, the computer program product including instructions configured to execute the system and / or perform a step or steps of the method of any of the disclosed embodiments. For example, in certain embodiments, the computer program product tangibly embodied in the non-transitory machine-readable recording medium includes instructions configured to cause one or more data processors to perform operations directing at least one of the following steps of determining methylation of the FMR1 gene in a sample from a subject: isolating total nucleic acid from the sample, the total nucleic acid including both methylated and unmethylated nucleic acid; contacting a first portion of the isolated total nucleic acid with a methyl-binding protein; isolating a portion of the unbound nucleic acid, thereby producing a nucleic acid fraction enriched for unmethylated nucleic acid; isolating the bound nucleic acid, thereby producing a nucleic acid fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acid; and determining the presence or absence of an FMR1 amplification product in both the fraction enriched for methylated nucleic acid and the fraction enriched for unmethylated nucleic acid. The computer program product may further include instructions for analyzing data from FRAX Me-PCR as disclosed in detail herein (see, eg, FIG. 6).

[0088] Thus, the computer program product may include instructions to perform the steps of providing the size and amount of each PCR amplicon from FRAX PCR and / or meFRAX PCR as a peak size and peak height. In certain embodiments, the analysis checks the sizing standard and peak height to ensure that the GS-PCR CE data quality is acceptable for analysis. The method may then use the GS-PCR peak data of the sample from FRAX PCR and / or meFRAX PCR to calculate the number of CGG repeats for each allele. Using these CGG repeat numbers, the method may then scan the GS-PCR CE data of the methylated DNA fraction in the HEX channel and the GS-PCR CE data of the unmethylated DNA fraction in the FAM channel, respectively, to identify corresponding peaks that are higher than the allele-specific peak height threshold. In certain embodiments, the methylation status calling algorithm does not identify additional peaks in the methylated or unmethylated fractions, other than the peaks detected using total DNA. In this manner, the CGG alleles detected by GS-PCR in the FRAX PCR assay are confirmed and their methylation status is determined. Detection of the same peak in total DNA and in either the methylated or unmethylated fraction, but not both, indicates a fully methylated or unmethylated state for the allele, respectively. If the same peak is present in both the methylated and unmethylated fractions and in total DNA, the allele is called as partially methylated. The system may further include instructions for reporting the results.

[0089] Thus, the systems and computer products may perform any of the methods or steps of those methods disclosed herein. One or more embodiments described herein may be implemented using program modules, engines, or components. A program module, engine, or component may include a program, subroutine, part of a program, or a software or hardware component that can perform one or more described tasks or functions. As used herein, a module or component may exist in a hardware component independent of other modules or components. Alternatively, a module or component may be a shared element or process of other modules, programs, or machines.

[0090] FIG. 8 shows a block diagram of an analysis system 800 used for detecting and / or quantifying progesterone metabolites. As shown in FIG. 8, one or more processor-executable modules, engines, or components (e.g., programs, codes, or instructions) can be used to execute various subsystems of the analyzer system according to various embodiments. The modules, engines, or components can be stored in a non-transitory computer medium. When needed, one or more of the modules, engines, or components can be loaded into a system memory (e.g., RAM) and executed by one or more processors of the analyzer system. In the example shown in FIG. 8, modules, engines, or components for executing the method of the present disclosure or for executing any of the systems of the present disclosure are shown.

[0091] Accordingly, Figure 8 illustrates an exemplary computing device 800 suitable for use in systems and methods according to the present disclosure. The exemplary computing device 800 includes a processor 805 that communicates with a memory 810 and other components of the computing device 800 using one or more communication buses 815. The processor 805 is configured to execute processor-executable instructions stored in the memory 810 to perform one or more methods or operate one or more stations to detect the methylation status of an FMR1 allele according to various examples (e.g., examples in Figures 1-7 or 9-11 or examples disclosed elsewhere herein). In this example, the memory 810 can store processor-executable instructions 825 that can analyze (820) results for the samples as discussed herein.

[0092] The computing device 800 in this example may also include one or more user input devices 830 (e.g., a keyboard, a mouse, a touch screen, a microphone, etc.) for receiving user input. The computing device 800 may also include a display 835 (e.g., a user interface) for providing visual output to a user. The computing device 800 may also include a communication interface 840. In some examples, the communication interface 840 may enable communication using one or more networks, including a local area network ("LAN"); a wide area network ("WAN") (e.g., the Internet); a metropolitan area network ("MAN"); a point-to-point connection or a peer-to-peer connection, etc. Communication with other devices may be accomplished using any suitable network protocol. For example, one suitable network protocol may include the Internet Protocol ("IP"), the Transmission Control Protocol ("TCP"), the User Datagram Protocol ("UDP"), or a combination thereof (e.g., TCP / IP or UDP / IP).

[0093] The disclosed methods, compositions, kits and system embodiments provide a high-throughput assay for characterization of the methylation status of FMR1 alleles in subjects in need thereof. Thus, in certain embodiments, the sensitivity and specificity of the analysis was >95%, or >98%, or 100%. Thus, in certain embodiments, the results of all samples in the methylation status call are made by the methylation status calling algorithm, and no false negative or false positive calls are made. Furthermore, the intra-assay and / or inter-assay reproducibility for the methylation status is >95%, or >98%, or 100%. Also, in certain embodiments, the FRAX mePCR methylation calling algorithm disclosed herein is 100% concordant with the calls made by manual analysis for the methylation status. The disclosed methods can provide semi-quantitative results. For example, in certain embodiments, the recommended DNA input for the FRAX mePCR assay is 1 μg, although higher or lower concentrations (e.g., about 0.2 μg) can be used. Furthermore, the performance of the assay remains robust for DNA extracted from blood specimens stored at 4°C for >60 days and buccal swab specimens stored at -20°C for >20 days.

[0094] The disclosed method, composition, kit and system significantly improve sample processing time and throughput.For example, up to 95 samples can be analyzed by the assay in 2 days and results can be provided.In contrast, one SB gel can analyze about 30 samples at one time (one gel), and it takes about one week to obtain final results. EXAMPLES

[0095] The present disclosure may be better understood with reference to the following non-limiting examples.

[0096] Example 1 - Determination of CGG repeats from total DNA by FRAX PCR The sample evaluation may involve a separate assay. 1. Gene-specific PCR to determine the length of the repeated CGG trinucleotide sequence in the FMR1 gene using primers outside the repeat region (FRAX-F1 and FRAX-R-6FAM). This is used to detect large CGG repeats. Products are sized according to number of base pairs using an ABI 3730xl Genetic Analyzer. 2. Non-anchored triplet-primed PCR to detect the expanded allele of the FMR1 gene. This PCR uses a primer that binds to the CGG region and one of the gene-specific PCR primers (FRAX-R-6FAM). 3. A gender-detecting PCR is performed for interpretation of the results by amplifying the XY homology region in the amelogenin gene, which results in an X-specific fragment of 212 bp and a Y-specific fragment of 218 bp.

[0097] The primers used for gene-specific PCR are shown in the table below. [Table 3]

[0098] More than 200 CGG repeats are considered as mutations. Premutations (PM) are from 55 CGG repeats to 200 CGG repeats. Peaks are identified by scanning for the first and second largest peaks (i.e., length of PCR product) greater than a given intensity (i.e., amount). More than 200 CGG repeats are considered as mutations. Premutations (PM) are from 55 CGG repeats to 200 CGG repeats.

[0099] Example 2 - Analysis of methylated versus unmethylated nucleic acids by meFRAX PCR This assay uses the same primer sequences and assay conditions used for the fragile X gene-specific PCR (GS-PCR) assay (Example 1) to identify the FMR1 CGG-rich promoter region. However, the methylation PCR assay differs from the GS-PCR assay in that unmethylated and methylated DNA are amplified in separate reactions such that the products are differentiated by the fluorescent dye used to label the reverse primer. The PCR products are then combined and separated by capillary electrophoresis. The presence or absence of GS-PCR products using methylated DNA and unmethylated templates indicates the methylation status of the corresponding FMR1 allele. See Tables 1 and 2 for the primers used.

[0100] Example 3 - Methods Whole blood, amniotic fluid, amniotic fluid cell culture, chorionic villus sampling, chorionic villus sample cell culture, saliva and buccal were identified as specimen types for this assay. Each clinical trial included one No Template Control (NTC) and three methylation controls (see below). To eliminate minor run-to-run variations in amplicon size fractionation of GS-PCR products by CE, especially for highly repetitive GC-rich regions that may ultimately affect methylation status calls, the FRAX mePCR assay is run on the same plate with total DNA as well as methylated and unmethylated DNA fractions for samples. This configuration also facilitates data visualization and analysis.

[0101] 1) Standardization of sample DNA For total DNA GS-PCR, DNA samples were diluted to 10 ng / μL in 10 mM Tris-HCl (pH 8.0). For enrichment of unmethylated and methylated DNA, DNA samples were normalized to 1 μg in 200 μL of 10 mM Tris-HCl (pH 8.0).

[0102] 2) Preparation of MBD-conjugated Protein A magnetic beads The MBD2a-Fc protein was mixed with Protein A magnetic beads for 15 minutes at room temperature on a tube rotator. The MBD2-Fc / Protein A magnetic beads mixture is stable at 4° C. for up to one week.

[0103] 3) Sonication Standardized DNA samples (1 μg) were sheared to 3 kb fragments using a Covaris S2 sonicator. The fragmented DNA samples were transferred to new tubes or plates. They may be stored at -20°C for up to 1 week.

[0104] 4) Enrichment of methylated / unmethylated DNA To each well of a 96-well plate, 20 μL of 1× Bind / Wash Reaction Buffer and 4 μL of MBD2-Fc / Protein A magnetic bead mixture were added, followed by 76 μL of fragmented DNA sample (total 100 μL). Immunoprecipitation of the methylated DNA was performed in a plate thermomixer at room temperature for 20 min. Once binding was complete, the beads were pulled down on a magnetic stand and the supernatants (which contained the unmethylated DNA fraction) were collected from each well and stored; the supernatants are stable at -20°C for up to 1 week. After three 3-min washes, the methylated DNA fractions from each sample were eluted with 30 μL of 10 mM Tris-HCl (pH 8.0) in a thermomixer at 65°C for 15 min. The samples were stable at -20°C for 1 week.

[0105] 5) GS-PCR amplification Amplification was performed in a 96-well PCR plate, which was divided equally into three parts. Columns 1-4, 5-8, and 9-12 were assigned to total DNA, unmethylated DNA, and methylated DNA, respectively, to test the same sample set in parallel. Controls were loaded into the first four wells, followed by clinical samples (run capacity = 28 samples + 4 controls). Four microliters of DNA sample (from steps 1 and 4) was added to 26 μL of PCR reaction cocktail. PCR conditions are shown in Table 4. PCR cycling time: 30 cycles, 4.5 hours. [Table 4]

[0106] 6) Capillary electrophoresis The long and short injections of the PCR products were separated on an ABI 3730xl fragment analyzer.

[0107] 7) Data Analysis The electrophoretic data were analyzed using GeneMapper v.4.0 software and a custom methylation state calling algorithm. 8) Assay controls 1) No template control (NTC): 10 mM Tris-HCl (pH 8.0) (cocktail blank) is used instead of the DNA sample to ensure the absence of reagent contamination throughout the process. 2) Unmethylated allele control (C1): GM20230 (Coriell; CDC genetic testing reference material (Amos Wilson et al., 2009, Consensus characterization of 16 FMR1 reference materials: a consortium study, J Mol Diagn., 2008;10:2-12), male cell line DNA carrying an allele with 53-55 CGG repeats. This allele is unmethylated and should be detected in both total and unmethylated DNA. 3) Fully methylated allele control (C2): GM09237 (Coriell), a male cell line DNA carrying an allele of 931-940 CGG repeats that is detected as an allele of >200 CGG repeats in both total and methylated DNA and is undetectable in the unmethylated DNA fraction. 4) Partially methylated allele control (C4): A blood DNA sample from a woman carrying an allele of normal repeat size. Both alleles are detected in total DNA, unmethylated DNA and methylated DNA.**

[0108] The methylation status calling algorithm produces two possible outcomes: (1) the methylation status of each allele, or (2) samples flagged for manual review (Table 5). [Table 5]

[0109] Example 4 - Confirmation A total of 26 samples were included in the confirmation study, consisting of five different sample types: six blood samples collected in either ACD (yellow top) or EDTA (light purple top) blood tubes, five amniotic fluid (AF) direct or culture samples, five chorionic villus sampling (CVS) direct or culture samples, five saliva samples, and five buccal swab samples. All of them had Southern blot (SB) results as confirmation for the methylation status call. The types and numbers of samples used for confirmation are listed in Table 6 below. In Table 5, PM = premutation and FM = full mutation. [Table 6]

[0110] The intra-assay reproducibility for methylation status calls by the FRAX mePCR calling algorithm was 100% based on 60 alleles. For inter-assay reproducibility, the methylation status calls were reproducible for 59 alleles (98.3%), with only one allele in one CVS replicate sample being called differently. Small allele size differences between replicates are expected due to experimental variability, especially for the highly repetitive GC-rich regions separated by CE. Furthermore, it was previously confirmed that the separation of CGG repeat numbers as determined by GS-PCR varies from 1 to 4 repeats depending on the length of the repeat. Thus, taking into account the inherent run-to-run variability, a difference of 1 to 2 repeats (i.e., 3 to 6 bases) between replicates is within the range of expected variability for normal or expanded alleles.

[0111] 1) Sensitivity and specificity of the analysis The sensitivity and specificity of the analysis were evaluated using 26 DNA samples, including 6 blood samples collected in either ACD (yellow top) or EDTA (light purple top) blood tubes, 5 amniotic fluid (AF) direct or culture samples, 5 chorionic villus sampling (CVS) direct or culture samples, 5 saliva samples, and 5 buccal swab samples.

[0112] The FRAX mePCR assay determined the methylation status of all alleles for all samples. All controls passed and no false positives or false negatives were called. The overall analytical sensitivity and specificity was 100% after manual review based on 98 alleles (including the mosaic samples). One additional allele in each of the two mosaic samples, AFC4 (Figure 8) and BL2 (Figure 9), had an inconclusive result by Southern analysis, and another mosaic sample, BL7 (Figure 10), was flagged for manual review by the methylation status calling algorithm.

[0113] For AFC4, the sample was not flagged for manual review because all alleles identified by GS-PCR using total DNA could be accounted for in the methylated and unmethylated fractions. However, when comparing the mePCR results with the SB data, we found that the allele at 75 CGGs, which was fully methylated based on the mePCR analysis (Figure 8), was not detected in the Southern blot (SB) assay as either the 3.0 kb or 5.4 kb bands corresponding to the unmethylated or methylated alleles, respectively. This allele appears to be a minor allele in the mosaic sample. Although it was identified by both GS-PCR and mePCR, its abundance in the sample is likely below the detection limit of autoradiography after overnight exposure. Importantly, the clinically significant methylation status of the fully mutated allele at >200 CGGs in AFC4 was clearly visible by both mePCR and SB analysis and was concordant between the two methods.

[0114] For BL2, the 163 CGG allele peak was detected in both the methylated and unmethylated DNA fractions (Figure 9). The SB assay also detected the methylated 163 CGG allele as a 5.7 kb band, but not the 3.3 kb band corresponding to the unmethylated allele. This is consistent with the detection by mePCR of a significantly higher peak in the methylated DNA fraction compared to the unmethylated DNA (11,975 rfu vs. 353 rfu), indicating that the signal for the unmethylated 163 CGG allele was likely below the detection limit of the SB assay. For either AFC4 or BL2, longer exposures were not available to conclusively determine the methylation status for any of these minor alleles.

[0115] The mosaic sample BL7, which was flagged for manual review, has extremely unstable alleles with a wide size range corresponding to approximately 110 CGG repeats to 175 CGG repeats. The tallest peak among the stutter peaks was 162 CGG repeats in size by GS-PCR and was detected only in the unmethylated fraction together with an allele of 78 CGG (Figure 10). The SB results showed a smear between 3.0 kb and 3.4 kb, which was confirmed after 3 days of autoradiography exposure and corresponded to an unmethylated allele with between 70 and 200 CGG repeats. The methylation status calling algorithm could not detect a peak higher than the 100 rfu threshold for an allele at >200 CGG repeats and correctly flagged the sample for manual review. Side-by-side visualization of electropherograms for total, methylated, and unmethylated PCR results found that the >200 CGG allele was observed only in the total and unmethylated DNA fractions, consistent with the Southern results. The unstable 162 CGG allele generated an unusually large number of stutter peaks during PCR amplification, which may have influenced the amplification efficiency of the abundant but less abundant FM allele in the unmethylated DNA fraction. DNA purification and concentration steps after enrichment of the unmethylated fraction helped to increase the FM allele signal to >100 rfu (Figure 10, unmethylated-enriched panel).

[0116] 2) DNA input tolerance Three blood DNA samples and one prenatal AFC DNA sample were run in this study and their concentrations were measured using a NanoDrop spectrophotometer. Three decreasing input amounts, from 1 μg, to 0.5 μg, to 0.2 μg, were tested in the assay and analyzed by the methylation status calling algorithm. The reduction in input to 0.2 μg still produced concordant calls for alleles that were either unmethylated or fully methylated, but some alleles that were partially methylated were poorly tolerated at the lower input amounts and produced discordant calls, especially at 0.2 μg.

[0117] We found that analysis of mePCR CE peak height data for the two alleles affected by lowering the input revealed that the peak heights in both DNA fractions decreased proportionally to the input, indicating the semi-quantitative capability of the assay. Notably, these alleles shared similar characteristics; i.e., the peak heights detected in either methylated (BL3-30rpt) or unmethylated (AFC2 FM allele) DNA were significantly lower than those in the other fraction. The signal difference between the two fractions could be very large, e.g., at least 10-fold difference at 1 μg input. Lowering the DNA input could easily reduce the peak height in the lower fraction below its call threshold, resulting in an inaccurate call. Since the "partially methylated" call is most affected regardless of its repeat size, a starting DNA input of 1 μg in the sonication step could be recommended for this FRAX mePCR assay.

[0118] 3) Sample stability tolerance Two whole blood specimens were stored at 4°C for at least 27 days and extracted at two time points, one within 7 days of receipt and one at least 27 days after receipt. Two buccal swab specimens stored at -20°C and extracted at two time points, one within 7 days and one after 20 days, were also included. We found that DNA extracted from blood samples stored up to 63 days did not adversely affect performance and produced the same allele size and methylation status calls as blood extracted within 7 days. Similarly, DNA extracted from Ultraflock (SWU19) or swabs (SWC17) stored at -20°C for 21 days produced identical results as DNA extracted immediately after collection. Thus, both sample types stored in the above conditions do not adversely affect assay performance.

[0119] 4) Maternal cell contamination (MCC) In this study, the methylation algorithm used allele sizes determined by GS-PCR using total DNA to identify corresponding peaks in the methylated and unmethylated CE data and make the methylation status calls.

[0120] 5) FRAX methylation calling algorithm test Methylation status analysis of FMR1 alleles in each sample was performed by determining the CGG repeat size in total DNA by GS-PCR. Once the allele repeat size was determined, the algorithm performs a basic search-and-match task using CE peak data generated using methylated and unmethylated DNA fractions from the same sample, as well as alleles identified by GS-PCR using total DNA, to determine the methylation status of each allele. Finally, the methylation status of each allele is called based on the criteria listed above (Table 5 - Methylation Status).

[0121] Based on the 26 samples tested in this study, the results from the FRAX mePCR methylation calling algorithm were 100% concordant with the results from the manual analysis. For further analysis of test concordance, 175 sample runs during assay development (including the same samples tested in separate runs) were also analyzed manually and with the above algorithm, and the results were 100% concordant between the two methods. Not only did the algorithm perform comparable to the manual analysis, it also performed as expected in identifying any samples whose input CE data quality was variable or whose genotype output was outside the normal range, and flagged those samples for "manual review."

[0122] 6) Report Typical FRAX mePCR results and interpretations are listed below in Tables 7 and 8. Final reporting will depend on the gender and number of CGG repeats per allele as determined by the FRAX PCR assay. Of note, for CVS, full mutations may not be methylated because methylation may not be fully established at the gestational age at which CVS is typically performed. [Table 7] [Table 8]

[0123] (Conclusion) The technical performance of the FRAX methylation PCR assay was confirmed to be reproducible and robust for blood DNA samples, prenatal DNA samples (amniotic fluid and chorionic villus sampling and their cultures), saliva DNA samples, and buccal DNA samples. 1) The sensitivity and specificity of the analysis was 100% based on 98 alleles, all of which were consistent with Southern blot results after manual review. All samples produced a methylation status call, and no false-negative or false-positive calls were made by the methylation status calling algorithm. Although the minor alleles in the two mosaic samples could not be conclusively resolved by Southern blot analysis, they were clearly detectable by the FRAX mePCR assay in total DNA and in either the methylated (AFC4) or unmethylated (BL2) DNA fractions. 2) Based on 10 specimens consisting of at least one blood, prenatal (cultured cells and direct), buccal, and saliva specimen type, the intra- and inter-assay reproducibility for methylation status was 100% (60 / 60 alleles) and 98.3% (59 / 60 alleles), respectively. One normal allele in the CVS replicate in the inter-assay run was called as partially methylated instead of unmethylated state, possibly due to sample collection and immature methylation in this tissue type, resulting in slight variation in peak height between runs when its peak height drifted around the peak calling threshold. 3) The recommended DNA input for the FRAX mePCR assay is 1 μg. Assay performance remained robust for DNA extracted from blood samples stored at 4°C for up to 63 days, as well as buccal swab samples stored at -20°C for 21 days. 4) The FRAX mePCR methylation calling algorithm was in 100% agreement with calls made by manual analysis of methylation status. 5) Not counting no-template controls, up to 95 samples can be analyzed and results provided in 2 days. One SB gel can analyze approximately 30 samples at a time (one gel), and if extended film exposure is required, it may take up to a week (at least 5-6 days) to obtain final results.

[0124] Example 5 - Exemplary embodiment As used below, any reference to methods or systems is to be understood disjunctively as a reference to each of those methods or systems (e.g., "exemplary embodiments 1-4 are to be understood as exemplary embodiments 1, 2, 3, or 4").

[0125] Exemplary embodiment 1 is a method for determining methylation of the FMR1 gene in a sample from a subject, the method comprising: isolating total nucleic acid from the sample, the total nucleic acid including both methylated and unmethylated nucleic acid; contacting a first aliquot of the isolated total nucleic acid with a methyl-binding protein; isolating a portion of the unbound nucleic acids, thereby producing a nucleic acid fraction enriched for unmethylated nucleic acids; isolating the bound nucleic acids, thereby producing a nucleic acid fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acid; and determining the presence or absence of FMR1 amplification products in both the fraction enriched for the methylated nucleic acids and the fraction enriched for the unmethylated nucleic acids. Includes.

[0126] Exemplary embodiment 2 is a method of any of the exemplary method embodiments above or below, the method comprising: determining the size of the FMR1 amplification product in at least one of the fraction enriched for the methylated nucleic acid and the fraction enriched for the unmethylated nucleic acid. Further includes:

[0127] Exemplary embodiment 3 is a method of any of the exemplary method embodiments above or below, the method comprising: determining the number of CGG repeats in the FMR1 gene for the amplification product in at least one of the fractions enriched for the methylated nucleic acid and / or the fractions enriched for the unmethylated nucleic acid. Further includes:

[0128] Exemplary embodiment 4 is a method of any of the exemplary method embodiments above or below, the method comprising: performing a PCR amplification of the FMR1 gene using a second aliquot of nucleic acid derived from the total nucleic acid and determining the size of at least one FMR1 amplified product. Further includes: Exemplary embodiment 5 is a method of any of the exemplary method embodiments above or below, the method comprising: determining the number of CGG repeats in the FMR1 gene for at least one amplification product obtained from the total nucleic acid fraction. Further includes:

[0129] Exemplary embodiment 6 is a method of any of the exemplary method embodiments described above or below, wherein determining a size of the FMR1 amplification product in at least one of the fractions enriched for the methylated nucleic acids and / or the fractions enriched for the unmethylated nucleic acids comprises comparing the size of the amplification product for the fractions enriched for the methylated nucleic acids and the fractions enriched for the unmethylated nucleic acids to a size of at least one FMR1 amplification product for the total nucleic acids.

[0130] Exemplary embodiment 7 is a method of any of the exemplary method embodiments described above or below, wherein the size of the FMR1 amplification product in at least one of the fraction enriched for the methylated nucleic acid and the fraction enriched for the unmethylated nucleic acid is determined to be the size of at least one FMR1 amplification product for the total nucleic acid.

[0131] Exemplary embodiment 8 is a method of any exemplary method embodiment described above or below, wherein at least one primer used to generate the FMR1 amplification product is labeled with a detectable moiety.

[0132] Exemplary embodiment 9 is a method of any of the exemplary method embodiments described above or below, wherein at least one primer used to generate an amplification product from the FMR1 gene from the fraction enriched for methylated nucleic acids and / or the fraction enriched for unmethylated nucleic acids and / or the total nucleic acids has the same sequence but is labeled with a different detectable moiety.

[0133] Exemplary embodiment 10 is a method of any exemplary method embodiment above or below, wherein the detectable moiety is a fluorescent dye.

[0134] Exemplary embodiment 11 is a method of any of the exemplary method embodiments described above or below, wherein the fluorescent dye used for detection of the amplification products from the fraction enriched for unmethylated nucleic acids and / or the total nucleic acids is 6-carboxyfluorescein (FAM) and the dye used for detection of the amplification products from the fraction enriched for methylated nucleic acids is hexachlorofluorescein (HEX).

[0135] Exemplary embodiment 12 is a method of any exemplary method embodiment described above or below, wherein the isolated total nucleic acid is fragmented into fragments having a size ranging from 2.4 kilobase pairs (kb) to 3.2 kilobase pairs (kb) prior to the step of contacting the isolated total nucleic acid with the methyl-binding protein.

[0136] Exemplary embodiment 13 is a method of any of the exemplary method embodiments described above or below, wherein the methyl-binding protein is a bifunctional polypeptide comprising (i) an Fc portion of an antibody, (ii) a short flexible peptide linker, and (iii) a DNA-binding domain of an MBD2 protein.

[0137] Exemplary embodiment 14 is a method of any of the exemplary method embodiments above or below, wherein the size of the FMR1 amplification product in the fraction enriched for the methylated nucleic acid and / or the fraction enriched for the unmethylated nucleic acid and / or the total nucleic acid is measured by capillary electrophoresis.

[0138] Exemplary embodiment 15 is a method of any of the exemplary method embodiments above or below, the method comprising: Determining whether the subject has either (i) partial methylation of FMR1; (ii) both methylated and unmethylated copies of FMR1; or (iii) complete methylation of FMR1. Further includes:

[0139] Exemplary embodiment 16 is a method of any of the exemplary method embodiments above or below, the method comprising: determining whether the subject has: (i) partial methylation of the expanded FMR1 allele; (ii) both a methylated and an unmethylated expanded FMR1 allele; or (iii) complete methylation of the expanded FMR1 allele. Further includes:

[0140] Exemplary embodiment 17 is a composition for carrying out any of the above or below described method embodiments.

[0141] Exemplary embodiment 18 is a composition of any of the exemplary composition embodiments described above or below, wherein the composition comprises at least one primer having the sequence of SEQ ID NO:1, 2, 3 or 4.

[0142] Exemplary embodiment 19 is a kit for carrying out any of the method embodiments described above or below, or for using any of the compositions of any of the composition embodiments described above or below.

[0143] Exemplary embodiment 20 is a kit of any of the exemplary kit embodiments described above or below, which kit includes at least one primer having the sequence of SEQ ID NO:1, 2, 3 or 4.

[0144] Exemplary embodiment 21 is a kit of any of the exemplary kit embodiments described above or below, which kit includes instructions for use.

[0145] Exemplary embodiment 22 is a system for carrying out any of the method embodiments described above or below, or for using any of the compositions of any of the composition embodiments described above or below, or for using any of the kits of any of the kit embodiments described above or below.

[0146] Exemplary embodiment 23 is a system of any of the exemplary system embodiments described above or below, the system comprising: components and / or stations for isolating total nucleic acid from said sample; components and / or stations for fragmenting the isolated total nucleic acid into specific size ranges; a component and / or station for contacting a first portion of the fragmented nucleic acid with a methyl-binding protein; components and / or stations for collecting a fraction of said nucleic acid that is enriched for methylated nucleic acids and a fraction of said nucleic acid that is enriched for unmethylated nucleic acids; components and / or stations for carrying out PCR amplification of the FMR1 gene separately for the nucleic acid enriched for the methylated nucleic acid and for the fraction of nucleic acid enriched for the unmethylated nucleic acid; and Components and / or stations for determining whether FMR1 gene sequences are present in the nucleic acid fraction enriched for its methylated nucleic acids and / or in the nucleic acid fraction enriched for its unmethylated nucleic acids, or both. Further includes:

[0147] Exemplary embodiment 24 is a system of any of the exemplary system embodiments described above or below, the system comprising: a component and / or station for determining the size of said FMR1 amplification product in at least one of said fractions enriched for said methylated nucleic acids and / or said fractions enriched for said unmethylated nucleic acids; and / or a component and / or station for determining the number of CGG repeats in the FMR1 gene for the amplification product in at least one of the fractions enriched for the methylated nucleic acid and / or the fractions enriched for the unmethylated nucleic acid; Further includes:

[0148] Exemplary embodiment 25 is a system of any of the exemplary system embodiments described above or below, the system comprising: a component and / or station for performing a PCR amplification of said FMR1 gene using an aliquot of nucleic acid derived from said total nucleic acid and determining the size of at least one FMR1 amplification product; Further includes:

[0149] Exemplary embodiment 26 is a system of any of the exemplary system embodiments described above or below, the system comprising: a component and / or station for determining the number of CGG repeats in the FMR1 gene for at least one amplification product obtained from the total nucleic acid fraction. Further includes:

[0150] Exemplary embodiment 27 is a system of any of the exemplary system embodiments described above or below, wherein the step of determining a size of the FMR1 amplification product in at least one of the fraction enriched for the methylated nucleic acids and the fraction enriched for the unmethylated nucleic acids comprises comparing the size of the amplification product for the fraction enriched for the methylated nucleic acids and / or the fraction enriched for the unmethylated nucleic acids to a size of at least one FMR1 amplification product for total nucleic acids.

[0151] Exemplary embodiment 28 is a system of any of the exemplary system embodiments described above or below, wherein the size of the FMR1 amplification product in at least one of the fraction enriched for the methylated nucleic acid and the fraction enriched for the unmethylated nucleic acid is determined to be the size of the size of at least one FMR1 amplification product for the total nucleic acid.

[0152] Exemplary embodiment 29 is a system of any of the above or below exemplary system embodiments, wherein at least one primer used to generate the FMR1 amplification product is labeled with a detectable moiety.

[0153] Exemplary embodiment 30 is a system of any of the exemplary system embodiments described above or below, wherein at least one primer used to generate an amplification product from the FMR1 gene from the fraction enriched for methylated nucleic acids and / or the fraction enriched for unmethylated nucleic acids and / or the total nucleic acids has the same sequence but is labeled with a different detectable moiety.

[0154] Exemplary embodiment 31 is a system of any of the above or below described exemplary system embodiments, wherein the detectable moiety is a fluorescent dye.

[0155] Exemplary embodiment 32 is a system of any of the exemplary system embodiments described above or below, wherein the fluorescent dye used for detection of the amplification products from the fraction enriched for unmethylated nucleic acids and / or the total nucleic acids is 6-carboxyfluorescein (FAM) and the dye used for detection of the amplification products from the fraction enriched for methylated nucleic acids is hexachlorofluorescein (HEX).

[0156] Exemplary embodiment 33 is a system of any of the exemplary system embodiments described above or below, wherein the isolated total nucleic acid is fragmented into fragments having a size ranging from 2.4 kilobase pairs (kb) to 3.2 kilobase pairs (kb) prior to the step of contacting the isolated total nucleic acid with the methyl-binding protein.

[0157] Exemplary embodiment 34 is a system of any of the above or below exemplary system embodiments, wherein the methyl-binding protein is a bifunctional polypeptide comprising (i) an Fc portion of an antibody, (ii) a short flexible peptide linker, and (iii) a DNA-binding domain of the MBD2 protein.

[0158] Exemplary embodiment 35 is a system of any of the exemplary system embodiments described above or below, wherein the size of the FMR1 amplification product in the fraction enriched for the methylated nucleic acid and / or the fraction enriched for the unmethylated nucleic acid and / or the total nucleic acid is measured by capillary electrophoresis.

[0159] Exemplary embodiment 36 is a system of any of the exemplary system embodiments described above or below, the system comprising: components and / or stations for determining whether the subject has either (i) partial methylation of FMR1; (ii) both methylated and unmethylated copies of FMR1; or (iii) complete methylation of FMR1. Further includes:

[0160] Exemplary embodiment 37 is a system of any of the exemplary system embodiments described above or below, the system comprising: components and / or stations for determining whether the subject has either (i) partial methylation of the expanded FMR1 allele; (ii) both a methylated and an unmethylated expanded FMR1 allele; or (iii) complete methylation of the expanded FMR1 allele. Further includes:

[0161] Exemplary embodiment 38 is a system of any of the exemplary system embodiments described above or below, the system comprising: A data processor and / or a non-transitory computer readable recording medium containing instructions. which instructions, when executed on one or more data processors, cause the one or more data processors to perform operations of the programmable software for controlling any of the stations and / or components of the system.

[0162] Exemplary embodiment 39 is a system of any of the exemplary system embodiments described above or below, the system comprising: A non-transitory computer readable recording medium containing instructions for analyzing data from FRAX PCR and / or FRAX me-PCR. Further includes:

[0163] Exemplary embodiment 40 is a system of any of the exemplary system embodiments described above or below, the system comprising: A non-transitory computer readable recording medium comprising instructions for carrying out the steps of providing the size and amount of each PCR amplicon from FRAX PCR (total nucleic acid) and FRAX mePCR as peak size and peak height. Further includes:

[0164] Exemplary embodiment 41 is a system of any of the exemplary system embodiments described above or below, where the analysis examines the sizing standards and peak heights to ensure that the GS-PCR CE data quality is acceptable for analysis.

[0165] Exemplary embodiment 42 is a system of any of the exemplary system embodiments described above or below, in which GS-PCR peak data of a sample derived from total DNA is used to calculate the number of CGG repeats for each allele.

[0166] Exemplary embodiment 43 is a system of any of the exemplary system embodiments described above or below, in which these CGG repeat numbers are used to scan the GS-PCR CE data of the methylated DNA fraction in the HEX channel and the GS-PCR CE data of the unmethylated DNA fraction in the FAM channel to identify corresponding peaks above an allele-specific peak height threshold, respectively.

[0167] Exemplary embodiment 44 is a system of any of the exemplary system embodiments described above or below, wherein the methylation state calling algorithm does not identify additional peaks in either the methylated or unmethylated fractions beyond those peaks detected using total DNA.

[0168] Exemplary embodiment 45 is a system of any of the exemplary system embodiments above or below, whereby, optionally, detection of the same peak in total DNA and in either the methylated or unmethylated fraction, but not both, indicates a fully methylated or unmethylated state, respectively, for that allele, and / or if the same peak is present in both fractions as well as in total DNA, the allele is called as partially methylated.

[0169] Exemplary embodiment 46 is a computer program product tangibly embodied in a non-transitory machine-readable recording medium, the computer program product including instructions configured to perform any of the method embodiments described above or below, or to use any of the compositions of any of the composition embodiments described above or below, or to use any of the kits of any of the kit embodiments described above or below, or to execute any of the components and / or stations of any of the composition embodiments described above or below.

[0170] Exemplary embodiment 47 is a computer program product of any of the exemplary computer program product embodiments described above or below, the computer program product comprising: determining the presence or absence of an FMR1 amplification product in both the nucleic acid fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids; determining a size of the FMR1 amplification product in at least one of the fraction enriched for the methylated nucleic acid and the fraction enriched for the unmethylated nucleic acid; comparing the size of the amplification products for the fraction enriched for the methylated nucleic acids and the fraction enriched for the unmethylated nucleic acids with the size of at least one FMR1 amplification product for the total nucleic acids; and determining the size of the FMR1 amplification product in at least one of the fractions enriched for the methylated nucleic acid and the fractions enriched for the unmethylated nucleic acid as being the size of at least one FMR1 amplification product for the total nucleic acid. Includes.

[0171] Exemplary embodiment 48 is a computer program product of any of the exemplary computer program product embodiments described above or below, the computer program product comprising: instructions configured to cause one or more data processors to perform operations directing at least one of the following steps: Including: isolating total nucleic acid from the sample, the total nucleic acid including both methylated and unmethylated nucleic acid; contacting a first aliquot of the isolated total nucleic acid with a methyl-binding protein; isolating a portion of the unbound nucleic acids, thereby producing a nucleic acid fraction enriched for unmethylated nucleic acids; isolating the bound nucleic acids, thereby producing a nucleic acid fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acid; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acid; and Determining the presence or absence of an FMR1 amplification product in both the fraction enriched for the methylated nucleic acids and the fraction enriched for the unmethylated nucleic acids.

[0172] Exemplary embodiment 49 is a computer program product of any of the exemplary computer program product embodiments described above or below, the computer program product comprising: A non-transitory computer readable recording medium containing instructions for analyzing data from FRAX PCR and / or FRAX me-PCR. Further includes:

[0173] Exemplary embodiment 50 is a computer program product of any of the exemplary computer program product embodiments described above or below, the computer program product comprising: A non-transitory computer readable recording medium comprising instructions for carrying out the steps of providing the size and amount of each PCR amplicon from FRAX PCR (total nucleic acid) and FRAX mePCR as peak size and peak height. Further includes:

[0174] Exemplary embodiment 51 is a computer program product of any of the exemplary computer program product embodiments described above or below, wherein the analysis examines the sizing standards and peak heights to ensure that the GS-PCR CE data quality is acceptable for analysis.

[0175] Exemplary embodiment 52 is a computer program product of any of the exemplary computer program product embodiments described above or below, wherein GS-PCR peak data of a sample derived from total DNA is used to calculate the number of CGG repeats for each allele.

[0176] Exemplary embodiment 53 is a computer program product of any of the exemplary computer program product embodiments described above or below, wherein using these CGG repeat numbers, the GS-PCR CE data of the methylated DNA fraction in the HEX channel and the GS-PCR CE data of the unmethylated DNA fraction in the FAM channel are each scanned to identify corresponding peaks above an allele-specific peak height threshold.

[0177] Exemplary embodiment 54 is a computer program product of any of the exemplary computer program product embodiments described above or below, wherein the methylation state calling algorithm does not identify additional peaks in either the methylated or unmethylated fractions beyond those peaks detected using total DNA.

[0178] Exemplary embodiment 55 is a computer program product of any of the exemplary computer program product embodiments described above or below, optionally wherein detection of the same peak in total DNA and in either the methylated or unmethylated fraction, but not both, indicates a fully methylated or unmethylated state for the allele, respectively, and / or if the same peak is present in both fractions as well as in total DNA, the allele is called as partially methylated.

[0179] Exemplary embodiment 56 is a method of any exemplary method embodiment described above or below, where the analysis examines the sizing standards and peak heights to ensure that the GS-PCR CE data quality is acceptable for analysis.

[0180] Exemplary embodiment 57 is a method of any of the exemplary method embodiments described above or below, wherein GS-PCR peak data of a sample derived from total DNA is used to calculate the number of CGG repeats for each allele.

[0181] Exemplary embodiment 58 is a method of any of the exemplary method embodiments described above or below, in which these CGG repeat numbers are used to scan the GS-PCR CE data of the methylated DNA fraction in the HEX channel and the GS-PCR CE data of the unmethylated DNA fraction in the FAM channel, respectively, to identify corresponding peaks above an allele-specific peak height threshold.

[0182] Exemplary embodiment 59 is a method of any exemplary method embodiment described above or below, wherein the methylation state calling algorithm identifies no additional peaks in either the methylated or unmethylated fractions beyond those peaks detected using total DNA.

[0183] Exemplary embodiment 60 is a method of any of the exemplary method embodiments described above or below, where, optionally, detection of the same peak in total DNA and in either the methylated or unmethylated fraction, but not both, indicates a fully methylated or unmethylated state for the allele, respectively, and / or if the same peak is present in both fractions as well as in total DNA, the allele is called as partially methylated.

Claims

1. 1. A method for determining methylation of the FMR1 gene in a sample from a subject, comprising: isolating total nucleic acid from the sample, wherein the total nucleic acid includes both methylated and unmethylated nucleic acid; contacting a first aliquot of the isolated total nucleic acid with a methyl-binding protein; isolating a portion of the unbound nucleic acids, thereby producing a nucleic acid fraction enriched for unmethylated nucleic acids; isolating the bound nucleic acids, thereby producing a nucleic acid fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for methylated nucleic acids; performing PCR amplification of the FMR1 gene using an aliquot of nucleic acid from the fraction enriched for unmethylated nucleic acid; and determining the presence or absence of an FMR1 amplification product in both the fraction enriched for said methylated nucleic acids and the fraction enriched for said unmethylated nucleic acids. A method comprising:

2. determining the size of the FMR1 amplification product in at least one of the fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids. The method of claim 1 further comprising:

3. determining the number of CGG repeats in the FMR1 gene for the amplification products in at least one of the fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids; The method of claim 1 further comprising:

4. performing PCR amplification of the FMR1 gene using a second aliquot of nucleic acid derived from the total nucleic acid and determining the size of at least one FMR1 amplification product. The method of claim 1 further comprising:

5. determining the number of CGG repeats in the FMR1 gene for at least one amplification product obtained from the total nucleic acid fraction; The method of claim 4 further comprising:

6. 3. The method of claim 2, wherein the step of determining the size of the FMR1 amplification product in at least one of the fractions enriched for methylated nucleic acids and the fractions enriched for unmethylated nucleic acids comprises comparing the size of the amplification product for the fraction enriched for methylated nucleic acids and / or the fraction enriched for unmethylated nucleic acids with the size of at least one FMR1 amplification product for total nucleic acids.

7. 7. The method of claim 6, wherein the size of the FMR1 amplification product in at least one of the fractions enriched for methylated nucleic acids and the fractions enriched for unmethylated nucleic acids is determined to be the size of at least one FMR1 amplification product for the total nucleic acids.

8. 10. The method of claim 1, wherein at least one primer used to generate the FMR1 amplification product is labeled with a detectable moiety.

9. 9. The method of claim 8, wherein the at least one primer used to generate an amplification product from the FMR1 gene from the fraction enriched for methylated nucleic acids and / or the fraction enriched for unmethylated nucleic acids and / or the total nucleic acids has the same sequence but is labeled with a different detectable moiety.

10. The method of claim 8 , wherein the detectable moiety is a fluorescent dye.

11. 11. The method of claim 10, wherein the fluorescent dye used for detection of the amplification products from the fraction enriched for unmethylated nucleic acids and / or from the total nucleic acids is 6-carboxyfluorescein (FAM), and the dye used for detection of the amplification products from the fraction enriched for methylated nucleic acids is hexachlorofluorescein (HEX).

12. 2. The method of claim 1, wherein the isolated total nucleic acid is fragmented into fragments having a size ranging from 2.4 kilobase pairs (kb) to 3.2 kilobase pairs (kb) prior to the step of contacting the isolated total nucleic acid with the methyl-binding protein.

13. 2. The method of claim 1, wherein the methyl-binding protein is a bifunctional polypeptide comprising (i) the Fc portion of an antibody, (ii) a short flexible peptide linker, and (iii) the DNA-binding domain of the MBD2 protein.

14. 3. The method of claim 2, wherein the sizes of the FMR1 amplification products in the fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids are measured by capillary electrophoresis.

15. determining whether the subject has either (i) partial methylation of FMR1; (ii) both a methylated and an unmethylated copy of FMR1; or (iii) full methylation of FMR1; and / or determining whether the subject has either (i) partial methylation of an expanded FMR1 allele; (ii) both a methylated and an unmethylated expanded FMR1 allele; or (iii) full methylation of an expanded FMR1 allele. The method of claim 1 further comprising:

16. determining the presence or absence of FMR1 amplification products in both the nucleic acid fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids; determining the size of the FMR1 amplification product in at least one of a fraction enriched for the methylated nucleic acid and a fraction enriched for the unmethylated nucleic acid; comparing the size of the amplification products for the fraction enriched for methylated nucleic acids and / or the fraction enriched for unmethylated nucleic acids with the size of at least one FMR1 amplification product for total nucleic acids; and determining the size of the FMR1 amplification product in at least one of the fraction enriched for the methylated nucleic acids and the fraction enriched for the unmethylated nucleic acids as being the size of at least one FMR1 amplification product for the total nucleic acids. The method of claim 1 further comprising:

17. A composition for carrying out the method of claim 1.

18. 18. The composition of claim 17, comprising at least one primer having the sequence of SEQ ID NO: 1 to 4.

19. A kit for carrying out the method of claim 1.

20. At least one primer having the sequence of SEQ ID NO: 1 to 4 Including, Positive and / or negative controls for assaying the methylation status of the FMRI gene and / or the FMR1 gene as appropriate, Instructions for use 20. The kit of claim 19, optionally comprising:

21. A system for performing the method of claim 1.

22. components for isolating total nucleic acids from said sample; a component for fragmenting the isolated total nucleic acid into specific size ranges; a component for contacting a first portion of the fragmented nucleic acid with a methyl-binding protein; a component for collecting a fraction of said nucleic acid that is enriched for methylated nucleic acids and a fraction of said nucleic acid that is enriched for unmethylated nucleic acids; Components for performing PCR amplification of the FMR1 gene separately for the fraction of nucleic acids enriched for methylated nucleic acids and the fraction of nucleic acids enriched for unmethylated nucleic acids; and Components for determining whether FMR1 gene sequences are present in the nucleic acid fraction enriched for the methylated nucleic acid and / or the nucleic acid fraction enriched for the unmethylated nucleic acid, or both.

22. The system of claim 21, further comprising:

23. 10. A computer readable medium containing instructions for performing the method of claim 1.

24. Instructions for carrying out a method for determining methylation of the FMR1 gene in a sample from a subject 24. The computer readable medium of claim 23, wherein the method comprises: determining the presence or absence of FMR1 amplification products in both the nucleic acid fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids; determining the size of the FMR1 amplification product in at least one of a fraction enriched for the methylated nucleic acids and a fraction enriched for the unmethylated nucleic acids; comparing the size of the amplification products for the fraction enriched for methylated nucleic acids and the fraction enriched for unmethylated nucleic acids with the size of at least one FMR1 amplification product for total nucleic acids; and determining the size of the FMR1 amplification product in at least one of the fraction enriched for the methylated nucleic acids and the fraction enriched for the unmethylated nucleic acids as being the size of the size of at least one FMR1 amplification product for the total nucleic acids.

1. A computer-readable medium comprising:

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