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WO2018107294A1 - Marqueurs de méthylation de l'adn pour troubles neuropsychiatriques et procédés, utilisations et kits associés - Google Patents

Marqueurs de méthylation de l'adn pour troubles neuropsychiatriques et procédés, utilisations et kits associés Download PDF

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Publication number
WO2018107294A1
WO2018107294A1 PCT/CA2017/051516 CA2017051516W WO2018107294A1 WO 2018107294 A1 WO2018107294 A1 WO 2018107294A1 CA 2017051516 W CA2017051516 W CA 2017051516W WO 2018107294 A1 WO2018107294 A1 WO 2018107294A1
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methylation
sample
profile
optionally
cpg loci
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PCT/CA2017/051516
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English (en)
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Rosanna WEKSBERG
Sanaa CHOUFANI
Darci BUTCHER
Michelle SIU
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The Hospital For Sick Children
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the disclosure relates to methods and kits for detecting and/or screening for a neuropsychiatric disorder or an increased likelihood or risk of a neuropsychiatric disorder (ND), in a human subject.
  • ND neuropsychiatric disorder
  • NDs Neuropsychiatric disorders
  • ASD autism spectrum disorder
  • ID intellectual disability
  • ADHD attention deficit/hyperactivity disorder
  • SZ schizophrenia
  • genetics plays an important etiologic role in these disorders.
  • the same genetic change can be associated with very broad neurodevelopmental, physiological and morphological phenotypes/outcomes. That is, genetics contributes only a fraction of the risk for a specific phenotype e.g. -25% in ASD.
  • the relative contributions of other factors such as environment and epigenetics, the latter of which refers to a mechanism of regulation of gene expression without altering the DNA sequence itself, are not well understood.
  • Heterogeneity, both genetic and phenotypic poses one of the greater challenges in understanding the underlying mechanisms of these disorders.
  • the present inventors assessed two types of genomic aberrations that have all been associated to an increased risk for neuropsychiatric disorders, including autism spectrum disorder (ASD) and others: 1 ) copy number variants (CNVs), which often involve large genomic regions and multiple genes, and 2) single gene mutations.
  • ASD autism spectrum disorder
  • CNVs copy number variants
  • Two genomic regions not known to contain genes that have confirmed and direct roles in epigenetic regulation, or epigenes, were chosen to examine the epigenetic outcomes that accompany these genomic alterations that are variably associated with neuropsychiatric disorders (NDs).
  • NDs neuropsychiatric disorders
  • mutations in one epigene were studied. This mutation and CNVs are associated with, but do not always present with neuropsychiatric outcomes including ASD, ID, ADHD, SZ or congenital anomalies.
  • the de novo gene mutation that was chosen causes haploinsuffiency in an epigene (chromodomain helicase DNA- binding protein 8 [CHD8]), and the two pathogenic CNVs that were selected are from genomic regions on chromosomes 16 (16p1 1 .2 deletions) and 22 (22q1 1 .2 deletions), which do not yet have a known direct effect on epigenes.
  • CCD8 chromodomain helicase DNA- binding protein 8
  • CHD8 mutations and 16p1 1 .2 deletions are the two most common single gene mutations and CNVs, respectively, associated with ASD, each accounting for no more than 1 % of ASD cases (Bernier, R. et al., 2014; Hanson, E. et al. , 2015).
  • 16p1 1 .2 deletions and duplications and 22q1 1 .2 deletions are also variably associated with ASD and/or SZ.
  • 22q1 1 .2 duplications generally have a milder phenotype, with evidence for protection against SZ risk. All 3 genomic aberrations are also associated with ID to varying degrees.
  • the present disclosure identifies DNA methylation (DNAm) markers which are capable of differentiating pathogenic 16p1 1 .2 or 22q1 1 .2 deletions or pathogenic CHD8 mutations from controls.
  • DNAm markers and the methods of their use described herein may provide useful alternative or supplementary diagnostics to currently available methods of detecting and/or screening for NDs, including autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), intellectual disability (ID) and schizophrenia (SZ), or likelihood of said NDs, because risk or increased likelihood of such disorders are known to be associated with these CNV deletions and the pathogenic mutation disclosed herein.
  • a method of detecting and/or screening for a neuropsychiatric disorder (ND), or an increased likelihood of an ND, in a human subject comprising determining a sample DNA methylation (DNAm) profile from a sample of DNA from said subject, said sample profile comprising:
  • the method further comprises determining the level of similarity of said sample profile to one or more control profiles, wherein (i) a high level of similarity of the sample profile to an ND specific control profile; (ii) a low level of similarity to a non-ND control profile; and/or (iii) a higher level of similarity to an ND specific control profile than to a non-ND control profile indicates the presence of, or an increased likelihood of, the ND.
  • the CpG loci of (a) comprise (i) CpG loci from Table 2 having an absolute delta-beta value > 0.05, optionally > 0.06, > 0.07, > 0.1 , or > 0.15; and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i).
  • the CpG loci of (b) comprise (i) CpG loci from Table 5 having an absolute delta-beta value > 0.05; and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i).
  • the CpG loci of (c) comprise (i) CpG loci from Table 8 having an absolute delta-beta value > 0.05, optionally > 0.06, > 0.07, > 0.1 , > 0.12, > 0.15 or > 0.17; and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i).
  • the ND may be any ND that is associated with the CNV 16p 1 .2 deletion with respect to the CpG loci of Table 2; may be any ND that is associated with the CNV 22q1 1 .2 deletion with respect to the CpG loci of Table 5 and/or may be any ND that is associated with a pathogenic CHD8 mutation with respect to the CpG loci of Table 8.
  • the ND may be autism spectrum disorder (ASD), schizophrenia (SZ), attention deficit/hyperactivity disorder (ADHD) or intellectual disability (ID).
  • the ND is autism spectrum disorder.
  • the ND is SZ.
  • the sample profile comprises CpG loci from a), b) and c).
  • a method of detecting and/or screening for a neuropsychiatric disorder (ND), or an increased likelihood of ND, in a sample from a human subject comprising:
  • a sample profile comprises CpG loci from (i), (ii) and (iii).
  • the CpG loci comprise CpG loci from Table 2 having an absolute delta-beta value > 0.05, optionally > 0.06, > 0.07, > 0.1 , or > 0.15.
  • the CpG loci comprise CpG loci from Table 8 having an absolute delta-beta value > 0.05, optionally > 0.06, > 0.07, > 0.1 , > 0.12, 0.15 or ⁇ 0.17.
  • the sample is a blood sample.
  • determining the sample methylation profile comprises the steps:
  • MS-SSCA methylation-sensitive single-strand conformation analysis
  • HRM high resolution melting analysis
  • COBRA combined bisulfite restriction analysis
  • MS-SnuPE
  • a higher level of similarity to the ND specific control profile than to the non-ND control profile, respectively, is indicated by a higher correlation value computed between the sample profile and the ND specific control profile than an equivalent correlation value computed between the sample profile and the non-ND control profile, optionally wherein the correlation value is a correlation coefficient.
  • the correlation coefficient is a linear correlation coefficient, optionally a Pearson correlation coefficient or a Spearman correlation coefficient.
  • a high level of similarity to the control profile is indicated by a Pearson correlation coefficient between the sample profile and the control profile having an absolute value between 0.5 to 1 , optionally between 0.75 to 1
  • a low level of similarity to the control profile is indicated by a correlation coefficient between the sample profile and the control profile having an absolute value between 0 to 0.5, optionally between 0 to 0.25.
  • the methylation level is measured as a ⁇ - value.
  • a ND Score is calculated according to following formula:
  • ND score(B) r (B, ND profile) - r (B, non-ND profile) where r is a Pearson correlation coefficient, and B is a vector of DNAm levels across the selected methylation loci in the sample, for example methylation loci listed in Tables 2, 5 or 8.
  • determining the sample methylation profile comprises contacting the DNA with at least one agent that provides for determination of a CpG methylation status of at least one, optionally all, of the selected CpG loci, wherein the agent comprises an oligonucleotide- immobilized substrate comprising a plurality of capture probes, each capture probe comprising a pair of capture oligonucleotides, wherein the capture oligonucleotide pairs comprise (a) an oligonucleotide comprising nucleotide sequence complementary to or identical to a nucleotide sequence of genomic DNA comprising a selected CpG, and (b) an oligonucleotide comprising nucleotide sequence complementary to or identical to a nucleotide sequence of genomic DNA comprising the same selected CpG locus of (a), in which the cytosine residue of the CpG locus is replaced with a thymine residue.
  • the contacting is under hybridizing conditions.
  • the methylation levels of the selected loci of at least one ND specific control profile is derived from one or more samples, optionally from historical methylation data for a patient or pool of patients, known to have a 16p1 1 .2 pathogenic deletion or duplication, a 22q1 1 .2 pathogenic deletion or duplication or a CHD8 pathogenic mutation. It is expected that subprofiles specific to certain ND phenotypes will be determined and used as a particular ND specific control profile.
  • the non-ND control profile comprises DNAm levels for the selected ND loci listed in Tables 2, 5 and/or 8.
  • the ND specific control profile comprises DNAm levels for the selected CpG loci listed in Tables 2, 5 and/or 8.
  • the methylation levels of associated CpG loci not listed in Tables 2, 5 and/or 8 is assumed to be equivalent to the methylation level of a CpG loci listed in Tables 2, 5 and/or 8 with which the CpG loci is associated.
  • the sample is derived from blood, fibroblast tissue, buccal tissue, lymphoblastoid cell line, saliva or a prenatal sample.
  • the prenatal sample is optionally a CVS, placenta, circulating fetal DNA and/or amniotic fluid sample.
  • the sample is derived from a tissue biopsy.
  • the sample is derived from blood.
  • the human subject is a fetus.
  • Another aspect provides a method of detecting and/or screening for neuropsychiatric disorders (ND), or an increased likelihood of ND, in a human subject, comprising determining a sample DNA methylation (DNAm) profile from a sample of DNA from said subject, said sample profile comprising:
  • the sample profile comprises genes from (a), (b) and (c).
  • the method further comprises determining the level of similarity of said sample profile to one or more control profiles, wherein (i) a high level of similarity of the sample profile to an ND specific control profile; (ii) a low level of similarity to a non-ND control profile; and/or (iii) a higher level of similarity to an ND specific control profile than to a non-ND control profile indicates the presence of, or an increased likelihood of, ND.
  • Another aspect of the disclosure provides a method of assigning a course of management for an individual with a neuropsychiatric disorder (ND), or an increased likelihood of ND, comprising:
  • the medical condition is selected from, but not limited to, developmental difficulties, ID and NDs, including neurodevelopmental disorders such as ASD and ADHD for both pathogenic CNVs and CHD8 mutations; for 16p1 1 .2 deletions/duplication- obesity, speech, impaired language and motor function, morphological anomalies (e.g. macro- and microcephaly); for 22q1 1 .2deletions/duplications- congenital anomalies (e.g. heart defects, cleft palate), impaired immune function; for both 16p1 1 .2 and 22q1 1 .2 CNVs, SZ, and seizures.
  • developmental difficulties, ID and NDs including neurodevelopmental disorders such as ASD and ADHD for both pathogenic CNVs and CHD8 mutations; for 16p1 1 .2 deletions/duplication- obesity, speech, impaired language and motor function, morphological anomalies (e.g. macro- and microcephaly); for 22q1 1 .2deletions/duplications- congenital anomalies (e.
  • kits for detecting and/or screening for a neuropsychiatric disorder (ND), or an increased likelihood of ND, in a sample comprising:
  • the kit further comprises bisulfite conversion reagents, methylation-dependent restriction enzymes, methylation-sensitive restriction enzymes, PCR reagents, probes and/or primers.
  • the probes or primers are specific to the selected CpG loci, selected from the loci in Tables 2, 5 and/or 8.
  • the kit further comprises a computer- readable medium that causes a computer to compare methylation levels from a sample at the selected CpG loci to one or more control profiles and computes a correlation value between the sample and control profile.
  • the computer readable medium obtains the control profile from historical methylation data for a patient or pool of patients known to have, or not have, one of the pathogenic deletions or mutation disclosed herein and often an associated ND, or known to have a particular methylation signature determined to be specific to a range of NDs as disclosed herein.
  • the computer readable medium causes a computer to update the control profile based on the testing results from the testing of a new patient.
  • Figure 1 shows a volcano plot showing the relationship between the average change in blood DNAm in the 16p1 1 .2 deletion cohort compared to normal controls ( ⁇ effect size, X-axis) and the statistical significance of such changes (p-value of the F-test implemented in the minfi software package, after Benjamini-Hochberg correction for multiple testing, shown in logarithmic scale, Y-axis).
  • Each semi-transparent point represents one of the CpG sites on the HumanMethylation450K array.
  • Array probes on sex chromosomes, near SNPs and cross-reactive probes were removed.
  • the vertical lines represent the effect size of 5% change in DNAm.
  • the data cohorts contained 8 affected samples and 20 normal controls.
  • Figure 2 shows hierarchical clustering of 8 affected samples (black bar) and 20 control samples (grey bar) from blood.
  • the heatmap shows the clustering based on the DNAm levels across the 91 CpG sites that exhibited significant changes in methylation (p ⁇ 0.05 and at least 5% DNAm difference) between the two cohorts. Clustering was performed based on the Pearson correlation metric with average linkage (correlation scale shown on the right).
  • Figure 4 shows a volcano plot showing the relationship between the average change in blood DNAm in the 22q1 1 .2 deletion cohort compared to normal controls ( ⁇ effect size, X-axis) and the statistical significance of such changes (p-value of the F-test implemented in the minfi software package, after Benjamini-Hochberg correction for multiple testing, shown in logarithmic scale, Y-axis).
  • Each semi-transparent point represents one of the CpG sites on the HumanMethylation450K array. Array probes on sex chromosomes, near SNPs and cross-reactive probes were removed.
  • the vertical lines represent the effect size of 5% change in DNAm.
  • the data cohorts contained 7 affected samples and 20 normal controls.
  • Figure 5 shows hierarchical clustering of 7 affected samples (black bar) and 20 control samples (grey bar) from blood.
  • the heatmap shows the clustering based on the DNAm levels across the 51 CpG sites that exhibited significant changes in methylation (p ⁇ 0.01 and at least 5% DNAm difference) between the two cohorts. Clustering was performed based on the Pearson correlation metric with average linkage (correlation scale shown on the right).
  • Figure 7 shows a volcano plot showing the relationship between the average change in blood DNAm in the CHD8 mutation cohort compared to normal controls ( ⁇ effect size, X-axis) and the statistical significance of such changes (p-value of the F-test implemented in the minfi software package, after Benjamini-Hochberg correction for multiple testing, shown in logarithmic scale, Y-axis).
  • Each semi-transparent point represents one of the CpG sites on the HumanMethylation450K array. Arrays probes on sex chromosomes, near SNPs and cross-reactive probes were removed.
  • the vertical lines represent the effect size of 5% change in DNAm.
  • the data cohorts contained 8 affected samples and 85 normal controls.
  • Figure 8 shows hierarchical clustering of 8 affected samples (black bar) and 85 control samples (grey bar) from blood.
  • the heatmap shows the clustering based on the DNAm levels across the 264 CpG sites that exhibited significant changes in methylation (p ⁇ 0.01 and at least 5% DNAm difference) between the two cohorts. Clustering was performed based on the Pearson correlation metric with average linkage (correlation scale shown on the right).
  • Figure 9 shows classification of various categories of blood DNAm samples.
  • Two median-methylation profiles were built over the 264 significant CpGs: one using the 8 mutation samples (circles), and another using the 85 Control samples (squares).
  • 4 variants were also classified, none of which showed a higher similarity to the pathogenic mutation cases, therefore classifying them as benign variants.
  • These 4 variants were known to harbour inherited, missense mutations and were therefore not expected to show higher similarity to the pathogenic mutation cases. Pearson correlation was used as the similarity metric.
  • CNVs copy number variants
  • isolated or “purified” when used in relation to a DNA molecule refers to a DNA molecule that is extracted and separated from one or more contaminants with which it naturally occurs.
  • methylation refers specifically to DNA methylation or DNAm, and more particularly to a modification in which a methyl group or hydroxymethyl group is added to the 5 position of a cytosine residue to form a 5-methyl cytosine (5-mCyt) or 5-hydroxymethylcytosine (5- hmC).
  • CpG locus or “methylation locus” refers to an individual CpG dinucleotide sequence in genomic DNA which is capable of being methylated.
  • Individual CpG loci may be identified by reference to an lllumina CpG locus (lllumina ID #) which is defined by a chromosome number, genomic coordinate (referenced to NCBI, hg19), genome build (37), and +/- strand designation to unambiguously define each CpG locus.
  • lllumina ID # lllumina CpG locus
  • the genomic information is publically available through the UCSC genome browser at https://genome.ucsc.edu/.
  • methylation level refers to a measure of the amount of methylation at a target site (for example, a CpG locus) within a DNA molecule in a sample.
  • the level of methylation can be measured for one or more CpG dinucleotides, or for a region of DNA. If the methylation level of a target site within a sample is higher than a reference level, the sample is considered to have increased methylation relative to the reference at the target site. Conversely, if the methylation level of a target site within a sample is lower than the reference level, the sample is considered to have a decreased methylation level relative to the reference at the target site.
  • the target site may be an individual CpG locus or a region of DNA comprising multiple CpG loci, for example, a gene promoter.
  • Methylation levels of a target site may be measured by methods known in the art, for example, as a " ⁇ value” or "beta value", which is calculated as:
  • ⁇ value intensity of the methylated target (M)/(intensity of the unmethylated target (U) + intensity of the methylated target (M) + 100)
  • a ⁇ value of zero indicates no methylation and a value of one indicates 100% methylation.
  • methylation status refers to whether a specified target DNA site is methylated or not methylated.
  • the target site may be an individual CpG locus or a region of DNA comprising multiple CpG loci, for example, a gene promoter.
  • a target site may have a methylation status of "methylated” or “hypermethylated” if the target has significantly higher methylation beta value in an ND specific control profile compared to a non-ND control profile.
  • a target site may have a methylation status of "not methylated” or “hypomethylated” if the target has significantly lower methylation beta value in an ND specific control profile compared to a non-ND control profile.
  • the methylation status of at least some CpG loci in a pathogenic duplication region of a CNV disclosed herein is likely to be opposite to that of the deletion, such that a locus that is hypermethylated in the deletion, is likely to be hypomethylated in a duplication.
  • delta beta or “delta ⁇ ” refers to the difference between the ⁇ value of a methylation target in two different samples, for example, the ⁇ value of a methylation target in an ND specific control profile and the ⁇ value of the same methylation target in a non-ND control profile.
  • the term "gene” refers to a genomic DNA sequence that comprises a coding sequence associated with the production of a polypeptide or polynucleotide product (e.g., rRNA, tRNA).
  • the methylation level of a gene as used herein encompasses the methylation level of sequences which are known or predicted to affect expression of the gene, including the promoter, enhancer, and transcription factor binding sites.
  • the term “enhancer” refers to a cis-acting region of DNA that is located up to 1 Mbp (upstream or downstream) of a gene.
  • sample methylation profile refers to the methylation levels at one or more target sequences in a subject's genomic DNA.
  • the target sequence may be an individual CpG locus or a region of DNA comprising multiple CpG loci, for example, a gene promoter or CpG island.
  • the methylation profile of a sample tested according to the methods disclosed herein is referred to as a sample profile.
  • the sample methylation profile is compared to one or more control profiles.
  • the control profile may be a reference value and/or may be derived from one or more samples, optionally from historical methylation data for a patient or pool of patients who are known to have (ND specific or positive control), or not have an ND (non-ND or negative control).
  • the patient or pool of patients is known to have a 16p1 1 .2 pathogenic deletion or duplication, a 22q1 1 .2 pathogenic deletion or duplication, or a pathogenic mutation in CHD8 and increased risk for NDs.
  • the historical methylation data can be a value that is continually updated as further samples are collected and individuals are identified as ND or not-ND or as individuals are identified with a particular ND phenotype.
  • the control profile represents an average of the methylation levels for selected CpG loci as described herein. Average methylation values may, for example, be the mean values or median values.
  • an "ND specific control profile” or “ND control profile” may be generated by measuring the methylation levels at specified target sequences in genomic DNA from an individual subject, or population of subjects, who are known to have a 16p1 1 .2 pathogenic deletion or duplication, a 22q1 1 .2 pathogenic deletion or duplication, or a CHD8 pathogenic mutation.
  • the ND specific control profile or ND control profile also includes the methylation levels at specified target sequences in genomic DNA from an individual subject, or population of subjects, who are known to have a particular ND, such as ASD, ADHD, SZ or ID, and a 16p1 1 .2 pathogenic deletion or duplication, a 22q1 1 .2 pathogenic deletion or duplication, or a CHD8 pathogenic mutation.
  • a "non-ND control profile" may be generated by measuring the methylation levels at specified target sequences in genomic DNA from an individual subject or population of subjects who are known to not have ND.
  • the tissue source from which the sample profile and control profile are derived is matched, so that they are both derived from the same or similar tissue.
  • the phrase "detecting and/or screening" for a condition refers to a method or process of determining if a subject has or does not have said condition. Where the condition is a likelihood or risk for a disease or disorder, the phrase “detecting and/or screening” will be understood to refer to a method or process of determining if a subject is at an increased or decreased likelihood for the disease or disorder.
  • sensitivity refers to the ability of the test to correctly identify those patients with the disease or disorder, such that a 100% sensitivity indicates a test that correctly identifies all patients with the disease or disorder. Sensitivity is calculated as:
  • Sensitivity (True Positives)/(True Positives + False Negatives).
  • a high sensitivity as used herein refers to a sensitivity of greater than 50%.
  • CpG or "CG” site refers to cytosine and guanosine residues located sequentially (5'->3') in a polynucleotide DNA sequence.
  • CpG island refers to a region of genomic DNA characterized by a high frequency of CpG sites, for example, a CpG island may be characterized by CpG dinucleotide content of at least 60% over the length of the island.
  • CpG island shore refers to a region of DNA occurring within 2kbp (upstream or downstream) of a CpG island.
  • body in reference to a gene refers to the genomic region covering the entire gene from the transcription start site to the end of the transcript.
  • distance from TSS refers to the genomic difference in base pairs between specific CpG locus and the nearest transcription start site.
  • a first CpG locus is "associated" with a second CpG locus, if the methylation status at the first locus is reasonably predictive of the methylation status of the second locus and vice versa.
  • CpG loci may be considered “associated”, for example, if they occur within the same CpG island, CpG island shore, gene promoter or gene enhancer region.
  • CpG loci may also be considered “associated” by virtue of their genomic proximity, for example, CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of each other may be considered associated.
  • the term "treating DNA from the sample with bisulfite” refers to treatment of DNA with a reagent comprising bisulfite, disulfite, hydrogen sulfite or combinations thereof, for a time and under conditions sufficient to convert unmethylated DNA cytosine residues to uracil, thereby facilitating the identification of methylated and unmethylated CpG dinucleotide sequences.
  • Bisulfite modifications to DNA may be detected according to methods known in the art, for example, using sequencing or detection probes which are capable of discerning the presence of a cytosine or uracil residue at the CpG site.
  • subject refers to a human subject and includes, for example, a fetus.
  • complementarity are used in reference to a first polynucleotide (which may be an oligonucleotide) which is in "antiparallel association" with a second polynucleotide (which also may be an oligonucleotide).
  • antiparallel association refers to the alignment of two polynucleotides such that individual nucleotides or bases of the two associated polynucleotides are paired substantially in accordance with Watson-Crick base-pairing rules.
  • Complementarity may be "partial,” in which only some of the polynucleotides' bases are matched according to the base pairing rules.
  • nucleic acid technology can determine duplex stability empirically by considering a number of variables, including, for example, the length of the first polynucleotide, which may be an oligonucleotide, the base composition and sequence of the first polynucleotide, and the ionic strength and incidence of mismatched base pairs.
  • hybridize refers to the sequence specific non- covalent binding interaction with a complementary nucleic acid.
  • Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C for 15 minutes, followed by a wash of 2.0 x SSC at 50°C for 15 minutes may be employed.
  • SSC sodium chloride/sodium citrate
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C for 15 minutes.
  • the temperature in the wash step can be at high stringency conditions, at about 65°C for 15 minutes.
  • At least moderately stringent hybridization conditions it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length.
  • the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature.
  • a 1 % mismatch may be assumed to result in about a 1 °C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% sequence identity, the final wash temperature will be reduced by about 5°C.
  • stringent hybridization conditions are selected.
  • Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C for 15 minutes. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1 -6.3.6 and in: Sambrook et al.
  • oligonucleotide refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • nucleic acid and/or “oligonucleotide” as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, and is intended to include DNA and RNA which can be either double stranded or single stranded, represent the sense or antisense strand.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof.
  • the term "amplify”, “amplifying” or “amplification” of DNA refers to the process of generating at least one copy of a DNA molecule or portion thereof.
  • Methods of amplification of DNA are well known in the art, including but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA) and rolling circle amplification (RCA).
  • CNV copy number variant
  • neuropsychiatric disorder refers to a neuropsychiatric disorder that has been associated with a 16p1 1 .2 pathogenic deletion or a duplication thereof, a 22q1 1 .2 pathogenic deletion or a duplication thereof and/or a CHD8 pathogenic mutation, and includes, without limitation, autism spectrum disorder (ASD) attention deficit hyperactivity disorder (ADHD), intellectual disability (ID), autistic symptoms and schizophrenia (SD). The subject may present with a combination of autism and/or one or more of these other neuropsychiatric disorders.
  • ASSD autism spectrum disorder
  • ADHD attention deficit hyperactivity disorder
  • ID intellectual disability
  • SD autistic symptoms
  • the subject may present with a combination of autism and/or one or more of these other neuropsychiatric disorders.
  • the term “16p1 1 .2” refers to the genomic region in which a recurrent deletion or reciprocal duplication occurs, affecting approximately 25 genes.
  • 16p1 1 .2 pathogenic deletion (OMIM# 61 1913) or "16p1 1 .2 pathogenic duplication” (OMIM# 614671 ) refers to aberrations in the gene dosage of these particular genomic regions due to deletions and duplications of ⁇ 600kb, where the majority of individuals harbouring this deletion or duplication will present with a range of CNV- specific phenotypes including morphological anomalies and NDs (Leung, T.Y. et al., 2010).
  • the term "22q1 1 .2" refers to the genomic region in which a recurrent deletion or reciprocal duplication occurs, affecting approximately 30-40 genes.
  • the term "22q1 1 .2 pathogenic deletion” (OMIM# 188400, 192430) or "22q1 1 .2 pathogenic duplication” (OMIM# 608363) refers to aberrations in the gene dosage of these particular genomic regions due to deletions or duplications of ⁇ 3Mb, where the majority of individuals harbouring this deletion or duplication will present with a range of CNV-specific phenotypes including morphological anomalies and NDs.
  • CHD8 refers to the chromodomain helicase DNA-binding protein 8 gene located in the 14q1 1 .2 region (OMIM# 610528).
  • CHD8 pathogenic mutation refers to loss of function mutations, including nonsense mutations and deletions, leading to a reduction of functioning protein product and associated with the observation of variable phenotypes including both morphological anomalies and NDs (e.g. ID, ADHD), in the majority if individuals harbouring theses mutations.
  • the instant disclosure identifies 91 distinct CpG loci, each of which show a statistically significant (corrected p-value ⁇ 0.05) difference in methylation levels between individuals with a 16p1 1 .2 pathogenic deletion and controls over the tested population.
  • the instant disclosure identifies 51 distinct CpG loci, each of which show as statistically significant (corrected p-value ⁇ 0.01 ) difference in methylation levels between individuals with a 22q1 1 .2 pathogenic deletion and controls over the tested population.
  • the instant disclosure identifies 264 CpG loci, each of which show as statistically significant (corrected p-value ⁇ 0.01 ) difference in methylation levels between individuals with a pathogenic CHD8 mutation with a high risk for ASD and non-ND controls over the tested population.
  • the methylation levels of the disclosed loci, or a subset thereof may be used in diagnostic testing for determining a pathogenic mutation associated with a genomic aberration such as a mutation that is associated with an increased risk for a neuropsychiatric disorder (ND), with up to 100% sensitivity and specificity.
  • CpG loci may be considered “associated”, for example, if they occur within the same CpG island, CpG island shore, gene promoter or gene enhancer region.
  • CpG loci may also be considered “associated” by virtue of their proximity, for example, CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of each other may be considered associated.
  • an aspect of the disclosure provides a method of detecting and/or screening for a neuropsychiatric disorder (ND), or an increased likelihood of ND, in a human subject, comprising determining a sample methylation profile from a sample of DNA from said subject, said sample profile comprising:
  • a non-limiting list of exemplary methods that may be used to determine methylation levels at a specified target sequence of DNA include: bisulfite sequencing, pyrosequencing, methylation-sensitive single-strand conformation analysis (MS-SSCA), high resolution melting analysis (HRM), methylation-sensitive single nucleotide primer extension (MS-SnuPE), base-specific cleavage/MALDI-TOF, methylation-specific PCR (MSP), methylation-sensitive restriction enzyme-based methods and/or microarray-based methods.
  • MS-SSCA methylation-sensitive single-strand conformation analysis
  • HRM high resolution melting analysis
  • MS-SnuPE methylation-sensitive single nucleotide primer extension
  • MSP methylation-specific PCR
  • methylation levels are measured using an agent that provides for determination of a CpG methylation status of at least one, optionally all, of the selected CpG loci, wherein the agent comprises an oligonucleotide-immobilized substrate comprising a plurality of capture probes, each capture probe comprising a pair of capture oligonucleotides, wherein the capture oligonucleotide pairs comprise (a) an oligonucleotide comprising nucleotide sequence complementary to or identical to a nucleotide sequence of genomic DNA comprising a selected CpG loci, and (b) an oligonucleotide comprising nucleotide sequence complementary to or identical to a nucleotide sequence of genomic DNA comprising the same selected CpG loci of (a), in which the cytosine residue of the CpG loci is replaced with a thymine residue.
  • an agent includes a "microarray", comprising
  • similarity of the DNAm profile from a sample to one or more control profiles may be used to identify individuals having a specific gene mutation or CNV, an ND, or an increased likelihood of ND or as not having an ND.
  • the method comprises determining the level of similarity of a sample profile to one or more control profiles, wherein (i) a high level of similarity of the sample profile to an ND specific control profile; (ii) a low level of similarity to a non-ND control profile; and/or (iii) a higher level of similarity to an ND specific control profile than to a non-ND control profile indicates the presence of, or an increased likelihood of, the ND.
  • control profile may be a reference value, or derived from one or more samples, optionally from historical methylation data for a patient or pool of patients.
  • the control profile may be a reference value and/or may be derived from one or more samples, optionally from historical methylation data for a patient or pool of patients who are known to have, or not have, a 16p1 1 .2 pathogenic deletion or duplication (for Table 2 loci), a 22q1 1 .2 pathogenic deletion or duplication (for Table 5 loci) or a CHD8 pathogenic mutation (for Table 8 loci) or from a patient or pool of patients who are known to have, ND and either a 16p1 1 .2 pathogenic deletion or duplication (for Table 2 loci), a 22q1 1 .2 pathogenic deletion or duplication (for Table 5 loci) or a CHD8 pathogenic mutation (for Table 8 loci).
  • the historical methylation data can be a value that is continually updated as further samples are collected and individuals are identified as ND or not- ND.
  • the control database may be stored on an online database, which is continually updated with methylation data from diagnosed ND and non- ND patients. It will be understood that the control profile represents an average of the methylation levels for selected CpG loci as described herein.
  • a reference value and/or a control profile from one or more samples, including historical methylation data may be derived from a subset of individuals that have a particular ND phenotype and either a 16p1 1 .2 pathogenic deletion or duplication (for Table 2 loci), a 22q1 1 .2 pathogenic deletion or duplication (for Table 5 loci) or a CHD8 pathogenic mutation (for Table 8 loci).
  • the "ND specific control profile" or “ND control profile” is generated by measuring the methylation levels at specified target sequences in genomic DNA from an individual subject, or population of subjects, who are known to have a 16p1 1 .2 pathogenic deletion or duplication, a 22q1 1 .2 pathogenic deletion or duplication, or a CHD8 pathogenic mutation.
  • the ND specific control profile or ND control profile also includes the methylation levels at specified target sequences in genomic DNA from an individual subject, or population of subjects, who are known to have a particular ND, such as ASD, ADHD, SZ or I D, and a 16p1 1 .2 pathogenic deletion or duplication, a 22q1 1 .2 pathogenic deletion or duplication, or a CHD8 pathogenic mutation.
  • the "non-ND control profile" is generated by measuring the methylation levels at specified target sequences in genomic DNA from an individual subject or population of subjects who are known to not have ND.
  • the tissue source from which the sample profile and control profile are derived is matched, so that they are both derived from the same or similar tissue.
  • the sample profile and control profile are derived from different tissues.
  • the ND specific control profile and the non-ND control profile are derived from historical data and can indicate similarity of a sample to either the ND or non-ND profiles.
  • Methods of determining the similarity between methylation profiles are well known in the art. Methods of determining similarity may in some embodiments provide a non-quantitative measure of similarity, for example, using visual clustering. In another embodiment, similarity may be determined using methods which provide a quantitative measure of similarity.
  • similarity may be measured using hierarchical clustering, optionally using Manhattan distance.
  • unsupervised hierarchical clustering of a sample with an ND specific control profile indicates similarity to the ND specific control profile.
  • unsupervised hierarchical clustering of a sample with a non-ND control profile indicates similarity to the non-ND control profile.
  • the Manhattan distance function computes the distance that would be traveled to get from one data point to the other if a grid-like path is followed.
  • the Manhattan distance between two items is the sum of the differences of their corresponding components.
  • n is the number of variables
  • Xi and Yi are the values of the variable, at points X and Y respectively.
  • similarity may be measured by computing a "correlation coefficient", which is a measure of the interdependence of random variables that ranges in value from -1 to +1 , indicating perfect negative correlation at -1 , absence of correlation at zero, and perfect positive correlation at +1 .
  • the correlation coefficient may be a linear correlation coefficient, for example, a Pearson product-moment correlation coefficient.
  • x and y are the beta values for various CpG loci in a sample profile and a control profile, respectively.
  • a correlation coefficient calculated between the sample profile and the control profile indicates a high level of similarity to the control profile when the correlation coefficient has an absolute value between 0.5 to 1 , optionally between 0.75 to 1 , and a low level of similarity to the control profile when the correlation coefficient has an absolute value between 0 to 0.5, optionally between 0 to 0.25.
  • any "correlation value" which provides a quantitative scaling measure of similarity between methylation profiles may be used to measure similarity.
  • a sample profile may be identified as belonging to an individual with an ND, or an increased likelihood of ND, where the sample profile has high similarity to the ND profile, low similarity to the non- ND profile, or higher similarity to the ND profile than to the non-ND profile.
  • a sample profile may be identified as belonging to an individual without an ND, or a decreased likelihood of ND, where the sample profile has high similarity to the non-ND profile, low similarity to the ND profile, or higher similarity to the non-ND profile than to the ND profile.
  • a sample profile may be identified as belonging to an individual with ND, or an increased likelihood of ND, based on calculation of an ND Score, which generally is defined by the following formula:
  • ND score(B) r (B, ND profile) - r (B, control profile)
  • r is the Pearson correlation coefficient
  • B is a vector of DNAm levels across the selected CpG loci.
  • a sample profile with a positive ND Score is more similar to the ND specific profile across the selected CpG loci, and is therefore classified as "ND"; whereas a sample with a negative ND Score is more similar to the non- ND profile across the selected CpG loci, and is classified as "not ND”.
  • sample refers to a biological sample comprising genomic DNA from a human subject.
  • the sample may, for example, comprise blood, fibroblast tissue, buccal tissue, and/or amniotic fluid.
  • the sample comprises blood.
  • ND and non-ND Median methylation levels for pathogenic mutation/CNV cases (ND or higher likelihood of ND) and benign (non-ND) cases reported in Tables 2, 5 and/or 8 were identified using whole blood samples. Based on DNAm profiles in other disorders with mutations in epigenes, it is predicted that the DNAm profile for ND and non-ND, can be present in other samples, for example, fibroblast tissue, buccal tissue, lymphoblastoid cell lines, saliva or a prenatal sample.
  • the prenatal sample is optionally a CVS, placenta, circulating fetal DNA and/or amniotic fluid sample.
  • Another aspect provides a method of detecting and/or screening for a neuropsychiatric disorder (ND), or an increased likelihood of an ND, in a human subject, comprising determining a sample DNAm profile from a sample of DNA from said subject, said sample profile comprising:
  • the method further comprises determining the level of similarity of said sample profile to one or more control profiles, wherein (i) a high level of similarity of the sample profile to an ND specific control profile; (ii) a low level of similarity to a non-ND control profile; and/or (iii) a higher level of similarity to an ND specific control profile than to a non-ND control profile indicates the presence of, or an increased likelihood of, the ND.
  • the genes comprise ANKMY1, CAPS2, CCDC17, GLIPR1L2 and IZUM01. It is shown in Table 3, for example, that at an absolute delta beta of 0.15 and p-value ⁇ 0.05, the 5 genes ANKMY1, CAPS2, CCDC17, GLIPR1L2 and IZUM01 provide a specificity of 100% and a sensitivity of 88%.
  • the genes comprise SOX9 and USP18. It is shown in Table 9, for example, that at an absolute delta beta of 0.15 and a p-value ⁇ 0.01 , Sox9 and USP18 provide a specificity of 65% and a sensitivity of 63%. [00111] It will also be appreciated by a person of skill in the art that the methods described herein can be used to distinguish between an ND with a pathogenic 16p1 1 .2 deletion, an ND with a 22q1 1 .2 pathogenic deletion and an ND with a CHD8 pathogenic mutation.
  • subsets of individuals with ND and a pathogenic 16p1 1 .2 deletion can be used to distinguish between subsets of individuals with ND and a pathogenic 16p1 1 .2 deletion, subsets of individuals with ND and a 22q1 1 .2 pathogenic deletion and subsets of individuals with ND and a CHD8 pathogenic mutation disclosed herein.
  • the subsets of individuals may be stratified based on ND phenotype, such as ASD, ADHD, ID or SZ.
  • a proper diagnosis of a particular ND genotype (which may, in some instances, be specific to a subset of ND phenotype) allows for testing, treatment and medical management appropriate for each condition, given the differences in their clinical characteristics.
  • Confirmation of a diagnosis of an ND aids in medical management by enabling targeted screening for the multisystem manifestations of these complex conditions, optimizing the opportunity for early intervention and management.
  • an aspect of the disclosure provides a method of assigning a course of management for an individual with a neuropsychiatnc disorder (ND), or an increased likelihood of an ND, comprising:
  • the term "a course of management” refers to any testing, treatment, medical intervention and/or therapy applied to an individual with an ND and/or symptoms of an ND.
  • Medical interventions include, but are not limited to, pharmaceutical treatments, surgical procedures, weight management, physical or occupational therapy.
  • behavioral and cognitive assessments, anticipatory follow up and guidance, monitoring and therapy would be indicated.
  • the medical condition associated with ND is selected from, but is not limited to, developmental delay, ASD, ID, ADHD, SZ, morphological anomalies (e.g. craniofacial, cardiac, etc.), obesity, and growth abnormalities.
  • kits for detecting and/or screening for a neuropsychiatnc disorder (ND), or an increased likelihood of an ND, in a sample comprising:
  • kits for detecting and/or screening for a neuropsychiatnc disorders (ND), or an increased likelihood of an ND, in a sample comprising: (a) at least one detection agent for determining:
  • the instructions for use comprise instructions for carrying out the methods disclosed herein.
  • the kit further comprises bisulfite conversion reagents, methylation-dependent restriction enzymes, methylation-sensitive restriction enzymes, PCR reagents, probes and/or primers.
  • the probes or primers are specific to selected CpG loci of Tables 2, 5 and/or 8.
  • the kit further comprises a computer- readable medium that causes a computer to compare methylation levels from a sample at the selected genes to one or more control profiles and compute a correlation value between the sample and control profile.
  • the kit further comprises a computer- readable medium that causes a computer to compare methylation levels from a sample at the selected CpG loci to one or more control profiles and compute a correlation value between the sample and control profile.
  • control profiles include profiles from individuals or reference values from individuals with ND and a pathogenic deletion or mutation disclosed herein.
  • the control profile is from individuals or reference values from individuals with a particular ND phenotype and a pathogenic deletion or mutation disclosed herein.
  • each epigenetic pattern is unique to each CNV and specific gene mutation but there is also further epigenetic variation likely due to each individual's genomic/environmental status. It is hypothesized that epigenetic patterns observed in association with specific CNVs can differentiate between highly prevalent phenotypes e.g. 16p .2 deletions with and without ASD. This approach can be applied in future to other genetically or clinically substratified groups, e.g.
  • variable SZ phenotype for the examination of the variable SZ phenotype observed with 22q1 1 .2 deletions.
  • the present inventors have demonstrated that epigenetic patterns specific to each mutation differentiates between pathogenic and benign mutations, allowing for more accurate clinical diagnosis where there are variable phenotypes presenting with the same genetic mutation. Given that many of these variable phenotypes overlap between specific gene mutations/CNVs, from a diagnostic or risk assessment perspective, it would be advantageous to be able to screen and categorize an individual using DNAm signatures through a DNA microarray, which is more cost and labour efficient than whole genome sequencing. These data can be adapted and utilized by clinicians to better classify patients molecularly and to assess for risk of various NDDs and neuropsychiatric conditions.
  • epigenes in the CNV region are not the only way in which epigenetic outcomes may be altered; other genes in the region, some of which have uncharacterized functions, may have downstream effects on epigenetic regulation, therefore also leading to aberrant DNAm profiles observed in association with the CNV.
  • genetic mutations in epigenes necessarily lead to epigenetic aberrations that can be implicated in the pathogenesis of a disorder.
  • 16p1 1 .2 pathogenic deletions and duplications exhibit some overlap as well as reciprocal and CNV-specific phenotypes given the differences in gene region dosage; seizures are observed with both the deletion and the duplication, most individuals with either CNV have speech and motor delay and dysmorphology, however ASD is more prevalent and more severe in the deletion, whereas congenital anomalies and ADHD are more common with the duplication (Fernandez, B.A. et al., 2010; Shinawi, M. et al., 2010). Macrocephaly is seen with the deletion, where the opposite phenotype, microcephaly, is observed with the duplication (Qureshi, A.Y. et al., 2014; McCarthy, S.E. et al., 2009). An increased risk for SZ (8-24-fold) is a neuropsychiatric outcome that is only seen with the duplication (McCarthy, S.E. et al., 2009).
  • the 16p1 1 .2 deletion signature was derived from a comparison of DNAm from 8 individuals with pathogenic deletions in the 600kb risk locus of the 16p1 1 .2 region to 20 controls (Table 2) using the analysis methodology previously described in the Methylation Array Analysis section.
  • the set of 91 probes for specific CpG sites comprising the 16p1 1 .2 deletion signature were located within the bodies or promoter regions of 49 known genes (Table 2). 12 individual genes had 2 or more differentially methylated probes, including GLI Pathogenesis-Related 1 Like 2 (GLIPR1L2, 9 probes), Eukaryotic Translation Initiation Factor 4E (EIF4E, 6 probes), and Coiled-Coil Domain Containing 17 (CCDC17, 4 probes).
  • the 16p1 1 .2 duplication signature is derived from a comparison of DNAm from individuals with pathogenic duplications in the 600kb risk locus of the 16p1 1 .2 region to control using the analysis methodology previously described in the Methylation Array Analysis section.
  • the LOO procedure confirms that a p-value threshold, when combined with an effect size threshold
  • is the necessary significance level at which the LOO procedure makes no classification errors (ie. Sensitivity and specificity of 100%).
  • is the necessary significance level at which the LOO procedure makes no classification errors (ie. Sensitivity and specificity of 100%).
  • a "signature set" of significant CpG sites is derived. It is expected that the signature set will likely contain some regions of methylation that will be opposite that of the deletion signature i.e., in certain regions where there is hypermethylation in the deletion signature, it is expected that there is hypomethylation in the duplication signature, and vice versa.
  • the set of probes for specific CpG sites comprising the 16p1 1 .2 duplication signature are located within the bodies or promoter regions of known genes.
  • the specificity of the signature CpGs is validated on a collection of normal blood samples derived from GEO. Similar to the LOO procedure, median DNAm profiles for the pathogenic 16p1 1 .2 duplication samples and for the control samples, respectively, are generated. The Pearson correlation of each of the GEO samples is computed with the reference 16p1 1 .2 duplication profile and the reference control profiles, using the significant CpGs sites. A high degree of specificity is achieved with the GEO control samples.
  • the signature is then applied to classify subjects with 16p 1 .2 duplications (including variable deletion sizes, variable phenotype) into either pathogenic or benign CNVs.
  • 22q1 1 .2 deletions are also strongly associated with, but do not always present with, ASD (-14% with ASD diagnosis, >20% with ASD symptoms) (Fine, S.E. et al., 2005), SZ (Murphy, K.C. et al., 1999) (20-25% of patients with deletions, but not with duplications, OMI M# 608363), as well as congenital heart malformations, cleft palate and T-cell immunodeficiency (Cohen, E. et al. , 1999).
  • the 22q1 1 .2 duplication typically has a milder phenotype, and therefore is largely undetected, but has a diverse range of phenotypes, ranging from normal phenotype to a combination of behavioural and morphological abnormalities (Yobb, T.M. et al. , 2005). It presents variably with seizures, heart defects and velopharyngeal insufficiency, examples of overlapping phenotypes with the deletion. There are also distinct phenotypes specific to the duplication including particular craniofacial abnormalities, growth and motor delay.
  • the 22q1 1 .2 deletion signature was derived from a comparison of DNAm from 7 individuals with pathogenic deletions in the 300Mb DiGeorge/Velocardiofacial Syndrome regions in the 22q1 1 .2 region to 20 controls (Table 5) using the analysis methodology previously described in the Methylation Array Analysis section.
  • the set of 51 probes for specific CpG sites comprising the 22q1 1 .2 deletion signature were located within the bodies or promoter regions of 42 known genes (Table 5). Only one gene contained multiple differentially methylated probes— 2 probes in the Developmental Pluripotency Associated 5 (DPPA5) gene.
  • the 22q1 1 .2 duplication signature is derived from a comparison of DNAm from individuals with pathogenic deletions in the 300Mb DiGeorge/Velocardiofacial Syndrome regions in the 22q1 1 .2 region to controls using the analysis methodology previously described in the Methylation Array Analysis section.
  • the LOO procedure confirms that a p-value threshold, when combined with an effect size threshold
  • the set of probes for specific CpG sites comprising the 22q1 1 .2 duplication signature are located within the bodies or promoter regions of known genes.
  • the signature is then applied to classify subjects with 22q1 1 .2 duplications (including variable deletion sizes, variable phenotype) into either pathogenic or benign CNVs.
  • CHD8 (14q1 1 .2, OMIM # 615032) belongs to the chromodomain helicase DNA binding (CHD) family of proteins, which use the energy from ATP hydrolysis to modify chromatin structure through alterations in nucleosome positioning and composition, thus modifying gene expression.
  • the CHD family is characterized by the presence of tandem chromo (chromatin organisation modifier) domains in the N-terminal region and a catalytic, central S/VF2-related helicase/ATPase domain (Tajul-Arifin, K. et al., 2003).
  • CHD8 is a known binding partner of CHD7, mutations in which lead to CHARGE syndrome (OMIM# 214800, coloboma of the eye, heart defects, choanal atresia, retardation of growth and development, genital hypoplasia, and ear/deafness/vestibular/olfactory/other cranial nerve disorders), which is another ASD-associated syndrome with a distinct methylation signature (Butcher, D.T. et al., 2013).
  • CHD8 plays a significant role during embryonic development by binding to ⁇ -catenin (Thompson, B.A. et al., 2008) and in its regulation of WNT signaling (Nishiyama, M. et al., 2012).
  • CHD8 mutations associated with ASD are rare, accounting for less than 1 % of ASD cases. Severe disruptive nonsense, splice and frameshift de novo mutations in CHD8 are highly associated with ASD (>85% of individuals), as well as other phenotypes including macrocephaly (80%), prominent supraorbital ridge (>75%), ID (60%), and attention problems (60%) (Bernier, R. et al. , 2014). Although nonsense mutations in CHD8 can be predictive of severity of outcome, the same cannot be said for other genetic variants such as missense mutations or variants of unknown significance (VUS). Therefore, additional molecular markers, such as of the presently disclosed DNAm signature, beyond that of genetics alone can help to properly classify mutations as pathogenic or benign.
  • the CHD8 signature was derived from a comparison of DNAm from 8 individuals with pathogenic mutations in the CHD8 gene to 85 controls (Table 8) using the analysis methodology previously described in the Methylation Array Analysis section.
  • the set of 264 probes for specific CpG sites comprising the CHD8 signature were located within the bodies or promoter regions of 173 known genes (Table 8). 32 individual genes had 2 or more differentially methylated probes, including Zinc Finger and BTB domain containing 22 (ZBTB22, 12 probes), CD47 antigen molecule (CD47, 5 probes) and ArfGAP with GTPase domain, Ankyrin repeat and PH domain 2 (AGAP2, 4 probes).
  • CHD8 is an ATP-dependent chromatin remodeling factor that regulates beta-catenin target genes. Mol Cell Biol 28, 3894-904 (2008). 35. Nishiyama, M., Skoultchi, A.I. & Nakayama, K.I. Histone H1 recruitment by CHD8 is essential for suppression of the Wnt-beta-catenin signaling pathway. Mol Cell Biol 32, 501 -12 (2012).

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Abstract

La présente invention concerne des signatures épigénétiques, comprenant des séquences dinucléotidiques CpG génomiques, des gènes et/ou des régions génomiques, qui sont méthylés de manière différentielle chez des individus présentant des variants de nombre de copies pathogènes ou une mutation pathogène qui sont associés à un trouble neuropsychiatrique (TN) ou à une probabilité accrue d'un TN et leur utilisation dans des procédés et des kits de détection et/ou de dépistage de TN ou de la probabilité d'un TN.
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