RELATED APPLICATION

[0001 ] This application claims the benefit of priority to United States Provisional Applications Nos. 62/065,374 filed October 17, 2014 and 62/076,765 filed November 7, 2014, respectively. The contents of which are incorporated herein by reference in their entirety.

FIELD

[0002] The disclosure relates to methods and kits for detecting and/or screening for Sotos syndrome (SS), or an increased likelihood of SS, in a human subject. The disclosure also relates to methods and kits for detecting and/or screening for Weaver syndrome (WVS), or an increased likelihood of WVS, in a human subject.

INTRODUCTION

[0003] Sotos syndrome (SS; OMIM 1 17550) is one of the most common and we!l-characterized overgrowth syndromes. It is characterized by pre- and postnatal overgrowth, advanced bone age, distinctive facial gestalt and a variety of neurodevelopmental problems including intellectual disability and behavioural difficulties [Sotos et ai, 1964]. Mutations in the nuclear receptor SET (su(var)3-9, enhancer-of-zeste, trithorax) domain containing protein-1 {NSD 1) gene are found in patients with Sotos syndrome [Baujat and Cormier-Daire 2007; Kurotaki et al, 2002; Tatton-Brown and Rahman 2007]. NSD1 is one of a large number of genes that developmental!y regulate the epigenome. NSD 1 encodes a histone lysine methyltransferase [Stec et al, 1998] and binds near various promoter elements to regulate transcription via interactions with H3K36 methylation and RNA polymerase II .

[0004] Weaver syndrome (WVS; OM IM 277590), first described in 1974, is an overgrowth syndrome characterized by tall stature, advanced bone age, characteristic facial features, variable intellectual disability and a range of associated clinical features including, poor coordination, soft doughy skin, camptodactyly of the fingers or toes, umbilical hernia, abnormal tone, and hoarse low cry [Weaver 1974; Tatton-Brown et al 2013]. Mutations in the enhancer of zeste (drosophila, homoiog 2) (EZH2) gene are found in patients with Weaver syndrome [Gibson et al 2012; Tatton-Brown et al 2011 ]. EZH2 encodes a histone methyltransferase involved in chromatin remodeling and transcriptional repression. EZH2 in combination with EED (Embryonic Ectoderm Development protein, mouse, homoiog of) and SUZ12 (Suppressor of Zeste 12, drosophila, homoiog of) forms the polycomb repressive complex (PRC2) which catalyzes the methylation of lysine residue 27 of histone 3 (H3K27).

[0005] Individuals with Weaver syndrome present with variable, somewhat non-specific clinical features that overlap with other overgrowth syndromes, most notably Sotos syndrome. Establishing a clinical diagnosis of Weaver syndrome is therefore challenging.

[0006] Targeted gene sequencing is the cornerstone of current molecular diagnostics for a multitude of genetic disorders, including Sotos syndrome. However, this diagnostic approach carries inherent challenges including inadequate power to identify all pathogenic mutations, in that the functional impact of non-synonymous variants (variants of unknown significance or "VUS") remains unclear. Several different pathogenicity prediction algorithms have been developed; however, these tools often provide incongruent results [Thusberg et al, 201 1]. Accordingly, there are a large number/percentage of cases for which the classification of single nucleotide variants into pathogenic and benign is problematic. Further, even when a pathogenic mutation is identified, little is known about the molecular and developmental pathophysiology of these disorders making it difficult to design targeted/specific therapies.

[0007] Accordingly, there is a need for robust and cost-effective tests capable of identifying Sotos syndrome and Weaver syndrome cases with high specificity and sensitivity. These tests may aiso be used to identify Sotos and Weaver syndrome in individuals carrying variants of unknown significance.

SUMMARY

[0008] The present disclosure provides DNA methylation markers which are capable of differentiating Sotos syndrome (SS) cases carrying a pathogenic NSD1 mutation from non-Sotos syndrome (non-SS) controls, including distinguishing Sotos syndrome cases from individuals carrying a benign NSD1 variant (benign variant as referred to herein means a variant in NSD1 gene that does not alter protein function). The DNA methylation 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 SS, or likelihood of SS.

[0009] In an aspect, there is provided a method of detecting and/or screening for Sotos syndrome (SS), or an increased likelihood of SS, in a human subject, comprising determining a sample DNA methylation profile from a sample of DNA from said subject, said sample profile comprising the methylation level of at least one, optionally at least 7, at least 10, at least 17, at least 25, at least 41 , at least 57, at least 87, at least 124, at least 156, at least 220, at least 287, at least 399, at least 549, at least 726, or all CpG loci from (i) Table 1 and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i).

[0010] 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 SS specific control profile; (ii) a low level of similarity to a non-SS control profile; and/or (iii) a higher level of similarity to a SS specific control profile than to a non-SS control profile indicates the presence of, or an increased likelihood of, SS.

[001 1] In an embodiment, the CpG loci comprise (i) CpG loci from Table 1 having an absolute SS delta-beta value≥ 0.4, optionally≥ 0.42, ≥ 0.44,≥ 0.46,≥ 0.5,≥ 0.52, > 0.54,≥ 0.56, > 0.58, > 0.6,≥ 0.62, > 0.64, or > 0.66; and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i).

[0012] In another aspect, there is provided a method of detecting and/or screening for Sotos syndrome (SS), or an increased likelihood of SS, in a human subject, comprising:

determining a sample methylation profile from a sample of DNA from said subject, said sample profile comprising the methylation level of CpG loci, wherein the CpG loci are the loci from Table 1 having an absolute SS de!ta-beta value≥ 0.4; and

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 SS specific control profile; (ii) a low level of similarity to a non-SS control profile; and/or (iii) a higher level of similarity to a SS specific control profile than to a non-SS control profile indicates the presence of, or an increased likelihood of, SS.

[0013] In an embodiment, the CpG loci comprise CpG loci from Table 1 having an absolute SS delta-beta value > 0.4, optionally≥ 0.42, > 0.44, > 0.46, > 0.48,≥ 0.5, > 0.52, > 0.54,≥ 0.56, > 0.58,≥ 0.6, ≥ 0.62,≥ 0.64 or > 0.66.

[0014] In another embodiment, determining the sample methylation profile comprises the steps:

a) providing the sample comprising genomic DNA from the subject;

b) optionally, isolating DNA from the sample;

c) optionally, treating DNA from the sample with bisulfite for a time and under conditions sufficient to convert non-methylated cytosines to uracils;

d) optionally, amplifying the DNA; and e) determining the methyiation level at the CpG loci by means of bisulfite sequencing, pyrosequencing, methylation-sensitive single-strand conformation analysis (MS-SSCA), high resolution melting analysis {HRM), combined bisulfite restriction analysis (COBRA), methylation-sensitive single nucleotide primer extension (MS-SnuPE), base-specific cleavage/MALDI- TOF, methylation-specific PCR (MSP), methylation-sensitive restriction enzyme-based methods, microarray-based methods, whole-genome bisulfite sequencing (WGBS, MethylC-seq or BS-seq), reduced-representation bisulfite sequencing (RRBS), and/or enrichment-based methods such as MeDIP-seq, MBD-seq, or MRE-seq.

[0015] In one embodiment, level of similarity is indicated by a correlation coefficient, in another embodiment, the correlation coefficient is a linear correlation coefficient, optionally a Pearson correlation coefficient or a Spearman correlation coefficient.

[0016] In another embodiment, 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 , and 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.

[0017] In another embodiment, a higher level of similarity to the SS specific control profile than to the non-SS control profile is indicated by a higher correlation value computed between the sample profile and the SS specific control profile than an equivalent correlation value computed between the sample profile and the non-SS control profile, optionally wherein the correlation value is a correlation coefficient, optionally a Pearson correlation coefficient or a Spearman correlation coefficient.

[0018] In an embodiment, the methyiation level is measured as a β- value. [0019] In another embodiment, a Sotos Syndrome Score (SS score) is calculated according to following formula:

SS score(B) - r (B, SS profile) - r (B, non-SS profile)

[0020] where r is a Pearson correlation coefficient, and B is a vector of DNA methylation levels across the selected CpG loci in the sample. In another embodiment, 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 ail, of the selected CpG loci, wherein the agent comprises an oligonucieotide-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.

[0021 ] In yet another embodiment, the contacting is under hybridizing conditions.

[0022] In an embodiment, the methylation levels of the selected loci of at least one control profile is derived from one or more samples, optionally from historical methylation data for a patient or pool of patients.

[0023] In another embodiment, the non-SS control profile comprises methylation levels for the selected CpG loci listed in Table 1. In yet another embodiment, the SS specific control profile comprises DNA methylation levels for the selected CpG loci listed in Table 1. In an embodiment, the methylation levels of associated CpG loci not listed in Table 1 is assumed to be equivalent to the methylation level of a CpG loci listed in Table 1 with which the CpG ioci is associated. [0024] In an embodiment, 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. In another embodiment, the sample is derived from a tissue biopsy.

[0025] In another embodiment, the human subject is a fetus.

[0026] Another aspect provides a method of detecting and/or screening for Sotos syndrome (SS), or an increased likelihood of SS, in a human subject, comprising determining the methylation level of at least one, optionally at least 6, at least 12, at least 17, at least 18, at least 24, at least 33, at least 49, at least 67, at least 72, at least 101 , at least 127, at least 164, at least 212, at least 275, or all the genes from Table 1 in a sample comprising genomic DNA from the subject, wherein a difference in methylation levels of genes from Table 1 , relative to a non-SS control profile is indicative of SS, or an increased likelihood of SS, in the subject.

[0027] Another aspect provides method of detecting and/or screening for Sotos syndrome (SS), or an increased likelihood of SS, in a human subject, comprising: determining a sample methylation profile from a sample comprising DNA from said subject, same sample profile comprising the methylation level of at least one, optionally at least 6, at least 12, at least 17, at least 18, at least 24, at least 33, at least 49, at least 67, at least 72, at least 101 , at least 127, at least 164, at least 212, at least 275, or all the genes from Table 1 ; and 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 a SS specific control profile; (ii) a low level of similarity to a non-SS control profile; and/or (ii) a higher level of similarity to a SS control profile than a non- SS control profile indicates the presence of, or an increased likelihood of, SS. [0028] In one embodiment, the methylation level is measured as a β- value.

[0029] In another embodiment, hypermethylation is indicated by the gene having a significantly higher methylation beta value in the SS specific control profile compared to the non-SS control profile and hypomethylation is indicated by the gene having a significantly lower methylation beta value in the SS specific control profile compared to the non-SS control profile.

[0030] in another embodiment, the sample is derived from blood, fibroblast tissue, buccal tissue, lymphoblastoid cell line, saliva or a prenatal sample, optionally a CVS, placenta, circulating fetal DNA and/or amniotic fluid sample.

[0031] In another embodiment, the human subject is a fetus.

[0032] Another aspect provides a method of assigning a course of management for an individual with Sotos syndrome (SS), or an increased likelihood of SS, comprising:

a) identifying an individual with SS or an increased likelihood of SS, according to the methods disclosed herein; and

b) assigning a course of management for SS and/or symptoms of a SS, comprising i) testing for at least one medical condition associated with SS and ii) applying an appropriate medical intervention based on the results of the testing.

[0033] In one embodiment, the medical condition is selected from congenita! heart defects, congenital renal abnormalities, hearing problems, vision problems, scoliosis, seizure activity, developmental/learning disabilities, behavioural problems and neuropsychological issues.

[0034] Another aspect of the disclosure provides a kit for detecting and/or screening for Sotos syndrome, or an increased likelihood of SS, in a sample, comprising:

(a) at least one detection agent for determining the methylation level of: i) at least one, optionally at least 7, at least 10, at least 17, at least 25, at least 41 , at least 57, at least 87, at least 124, at least 156, at least 220, at least 287, at least 399, at least 549, at least 726, or all CpG loci selected from Table 1 , and/or

ii) at least one, optionally at least 6, at least 12, at least 17, at feast 18, at least 24, at least 33, at least 49, at least 67, at least 72, at least 101 , at least 127, at least 164, at least 212, at least 275, or all the genes from Table 1 ; and

(b) instructions for use.

[0035] In an embodiment, the kit further comprises bisulfite conversion reagents, methylation-dependent restriction enzymes, methylation-sensitive restriction enzymes, PCR reagents, probes and/or primers.

[0036] In an embodiment, 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. In an embodiment, 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, Sotos syndrome. In some embodiments, the computer readable medium causes a computer to update the control profile based on the testing results from the testing of a new patient.

[0037] The present disclosure also provides DNA methylation markers which are capable of differentiating Weaver syndrome (WVS) cases carrying a pathogenic EZH2 mutation from non-Weaver syndrome (non-WVS) controls, including distinguishing Weaver syndrome cases from individuals carrying a benign EZH2 variant (benign variant as referred to herein means a variant in EZH2 gene that does not alter protein function). The DNA methylation 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 WVS, or likelihood of WVS.

[0038] Accordingly, another aspect of the disclosure provides a method of detecting and/or screening for Weaver syndrome (WVS), or an increased likelihood of WVS, in a human subject, comprising: determining a sample methylation profile from a sample comprising DNA from said subject, said sample profile comprising the methylation level of at least one, optionally at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50 or all CpG loci from (i) Table 8 and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i); and

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 WVS specific control profile; (ii) a low level of similarity to a non-WVS control profile; and/or (iii) a higher level of similarity to a WVS specific control profile than to a non-WVS control profile indicates the presence of, or an increased likelihood of, WVS.

[0039] In one embodiment, determining the sample methylation profile comprises the steps:

a) providing the sample comprising genomic DNA from the subject;

b) optionally, isolating DNA from the sample;

c) optionally, treating DNA from the sample with bisulfite for a time and under conditions sufficient to convert non-methylated cytosines to uracils;

d) optionally, amplifying the DNA; and

e) determining the methylation level at the CpG loci by means of bisulfite sequencing, pyrosequencing, methylation-sensitive single-strand conformation analysis (MS-SSCA), high resolution melting analysis (HRM), combined bisulfite restriction analysis (COBRA), methylation-sensitive single nucleotide primer extension (MS-SnuPE), base-specific cleavage/MALDI- TOF, methylation-specific PCR (MSP), methylation-sensitive restriction enzyme-based methods, microarray-based methods, whoie-genome bisulfite sequencing (WGBS, MethylC-seq or BS-seq), reduced-representation bisulfite sequencing(RRBS), and/or enrichment-based methods such as MeDIP-seq, MBD-seq, or MRE-seq.

[0040] In another embodiment, a high 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 0.5 to 1 , optionally between 0.75 to 1 , and 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.

[0041 ] In another embodiment, the level of similarity to the VWS specific profile than to the non-WVS control profile is indicated by a higher correlation value computed between the sample profile and the WVS specific profile than an equivalent correlation value computed between the sample profile and the non-WVS control profile, optionally wherein the correlation value is a correlation coefficient.

[0042] In another embodiment, the correlation coefficient is a linear correlation coefficient, optionally a Pearson correlation coefficient.

[0043] I n another embodiment, methylation level is measured as a β- value.

[0044] In another embodiment, a Weaver Syndrome Score (WVS score) is calculated according to following formula:

WVS score(B) = r (B, WVS profile) - r (B, non-WVS profile) where r is a Pearson correlation coefficient, and B is a vector of DNA methy!ation levels across the selected CpG loci in the sample.

[0045] In another embodiment, determining the profile of methylated DNA from the subject comprises contacting the DNA with at least one agent that provides for determination of a CpG methyiation 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 nucieotide 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.

[0046] Optionally, the contacting is under hybridizing conditions,

[0047] In another embodiment, the methyiation levels of the selected loci of at least one control profile is derived from one or more samples, optionally from historical methyiation data for a patient or pool of patients.

[0048] In another embodiment, the non-WVS control profile comprises methyiation levels for the selected CpG loci listed in Table 8.

[0049] in another embodiment, the WVS specific control profile comprises methyiation levels for the selected CpG loci listed in Table 8.

[0050] Optionally, the methyiation level of a selected CpG locus not listed in Table 8 is assumed to be equivalent to the methyiation level of a CpG locus listed in Table 8 with which the selected DNA CpG locus is associated.

[0051 ] In another embodiment, the sample is derived from blood, fibroblast tissue, buccal tissue, lymphoblastoid cell line, saliva or a prenatal sample, optionally a CVS, placenta, circulating fetal DNA and/or amniotic fluid sample.

[0052] In yet another embodiment, the human subject is a fetus.

[0053] Another aspect of the disclosure provides a method of detecting and/or screening for Weaver syndrome (WVS), or an increased likelihood of WVS, in a human subject, comprising: determining a sample methylation profile from a sample comprising DNA from said subject, same sample profile comprising the methylation level of at least one, optionally at least 2, at least 4, at least 6, or all the genes from Table 8; and 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 a WVS specific control profile; (ii) a low level of similarity to a non-WVS control profile; and/or (ii) a higher level of similarity to a WVS control profile than a non-WVS control profile indicates the presence of, or an increased likelihood of, WVS.

[0054] In one embodiment, determining the methylation levels of the selected genes comprises the steps:

a) providing the sample comprising genomic DNA from the subject;

b) optionally, isolating DNA from the sample; c) optionally, treating DNA from the sample with bisulfite for a time and under conditions sufficient to convert non-methylated cytosines to uracils;

d) optionally, amplifying the DNA; and

e) determining the methylation status at the selected genes by means of bisulfite sequencing, pyrosequencing, methylation-sensitive single-strand conformation analysis (MS-SSCA), high resolution melting analysis (HRM), combined bisulfite restriction analysis (COBRA), methylation- sensitive single nucleotide primer extension (MS-SnuPE), base-specific cleavage/MALD!-TOF, methylation-specific PCR (MSP), methylation-sensitive restriction enzyme-based methods, microarray-based methods, whole- genome bisulfite sequencing (WGBS, MethylC-seq or BS-seq), reduced- representation bisulfite sequencing (RRBS), and/or enrichment-based methods such as MeDIP-seq, MBD-seq, or MRE-seq.

[0055] In another embodiment, the methylation level is measured as a β-value.

[0056] In another embodiment, hypermethylation is indicated by the selected gene having a significantly higher methylation beta vale in the WVS specific control profile compared to the non-WVS control profile and hypomethylation is indicated by the selected gene having a significantly lower methylation beta value in the WVS specific control profile compared to the non-WVS control profile.

[0057] In another embodiment, the sample is derived from blood, fibroblast tissue, buccal tissue, lymphoblastoid cell line, saliva or a prenatal sample, optionally a CVS, placenta, circulating fetal DNA and/or amniotic fluid sample.

[0058] In another embodiment, the human subject is a fetus.

[0059] The present disclosure also provides a method of determining a course of management for an individual with Weaver syndrome (WVS), or an increased likelihood of WVS, comprising:

a) identifying an individual with WVS or an increased likelihood of WVS, according to the methods described herein; and

b) assigning a course of management for WVS and/or symptoms of a WVS, comprising i) testing for at least one medical condition associated with SS and ii) applying an appropriate medical intervention based on the results of the testing. [0060] In one embodiment, the medical condition is selected from scoliosis, camptodactyly, tumor growth, developmental/learning disabilities, intellectual disabilities, hypotonia and ligamentous laxity.

[0061 ] The present disclosure also provides a kit for detecting and/or screening for Weaver syndrome, or an increased likelihood of VWS, in a sample, comprising:

a) at least one detection agent for determining the methylation level of (i) at least one, optionally at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or all CpG loci from Table 8, and/or (ii) at least one, optionally at least 2, at least 4, at least 6, or ail the genes from Table 8; and

b) instructions for use.

[0062] In one embodiment, the kit further comprises bisulfite conversion reagents, methylation-dependent restriction enzymes, methylation-sensitive restriction enzymes, PCR reagents, probes and/or primers.

[0063] In yet another embodiment, 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.

DRAWINGS

[0064] Embodiments are described below in relation to the drawings in which:

[0065] Figure 1 identifies a large number of differentially methylated CGs between SS and control subjects. The volcano plot displays the relationship between difference in DNAm levels and significance between the two groups, using a scatter plot view. The y-axis is the negative log 10 of P values (a higher value indicates greater significance) and the x-axis is the difference in DNAm between SS and Control (non-SS) subjects. Light grey horizontal line depicts significance level at p<0.05. Note that most of the significant CpGs are losing methylation in SS compared to non-SS. At 20% cut-off difference in DNA methylation, it is possible to clearly enrich for the highly significant CpGs with larger difference between SS and non-SS.

[0066] Figure 2 shows a DNA methylation signature associated with NSD1 mutations. Unsupervised Hierarchical clustering of 72 samples based on the identified 7K differentially methylated CpG sites. All SS samples clustered together separate from non-SS control blood samples.

[0067] Figure 3 depicts testing of the sensitivity and the specificity of the NSD1 ^+/- DNAm signature. Using 19 SS and 53 non-SS subjects, the median-methylation profiles of SS and non-SS controls were generated, respectively, on the 7K CG sites. The specificity of these profiles were tested on a test set of GEO blood samples (n=1056, light grey filled circles), all of which were more similar to the non-SS control profile (specificity 100%). The sensitivity was also estimated on a separate SS test set (n=19, represented as squares), all of which were more similar to the SS profile than to the non- SS control (100% sensitivity). Similarity was computed as the Pearson correlation to either the SS or the non-SS control DNAm profile. Out of 16 missense samples (represented as X), 9 classify with SS and 7 with controls. Also shown the classifications of the original 53 controls (represented as triangles) and 19 SS (represented as dark grey filled circles) used in the derivation of the SS and non-SS control signatures.

[0068] Figure 4 shows that a NSD1 ^+/- DNAm signature can be identified in SS fibroblasts. Unsupervised Hierarchical clustering of 3 SS derived fibroblasts and 4 non-SS control derived fibroblasts clearly distinguish SS from control. In addition, tissue-specific DNAm is identified in fibroblast samples compared to controls.

[0069] Figure 5 depicts a series of leave-one-out (LOO) cross- validations on the combined dataset of 47 Sotos cases (38 original SS cases + 9 pathogenic missense cases) vs. 53 controls. At each LOO iteration, one sample was removed from the dataset for the subsequent validation step. The remaining samples were used to generate median DNA methylation profiles for the SS group and for the non-SS group. This classification was compared to the sample's true status (SS or non-SS). Based on these LOO results over all 47+53 samples, the specificity and sensitivity were estimated for various threshold combinations of delta beta (from 40% to 70%). In a second set of validations, an SS signature was created for different parameter combinations, and 1056 normal controls from the GEO collections were classified based on their closeness to SS or non-SS profiles. The number of samples correctly classified as "non-SS" contributed to the specificity estimate.

[0070] Figure 6 shows unsupervised hierarchical clustering of the EZH2+/- DNA methylation signature. Differential DNA methylation analysis using ttest statistics and false discovery rate between 7 subjects with EZH2+/- single nucleotide variants (or missense mutations) and 52 control subjects identified 3216 CpG sites to be significantly different. Unsupervised hierarchical clustering using Euclidian distance metrics identified a subset of 6 subjects with EZH2+/- that clustered together separate of all control samples and one subject with EZH2+/- (Subject F0123) that clustered with the control samples.

[0071 ] Figure 7 shows a principal component analysis revealing a clear separation between EZH2+/- and the controls,

[0072] Figure 8 shows a principal component analysis for a minimal EZH2+/- signature.

[0073] Figure 9 shows unsupervised hierarchical clustering of CpG sites showing the separation between Weaver syndrome and control samples.

[0074] Figure 10 shows a gene cluster on chromosome 7 is regulated by EZH2. A chromosome-wide view of the DNA methylation difference between Weaver syndrome and control samples shows the mapping of the genomic region on the short arm of chromosome 7 overlapping the HOXA gene cluster.

DESCRIPTION OF VARIOUS EMBODIMENTS

[0075] The inventors have conducted genome-wide DNA methylation (DNAm) profiling using blood from individuals with Sotos syndrome (SS), a disorder involving aberrant NSD1 function. Based on comparison of the DNA methylation profile from SS individuals to those of non-SS controls, the inventors have shown that DNA methylation profiles may be used in a test for early and accurate diagnosis of Sotos syndrome due to NSD1 pathogenic mutations. 7085 CpG loci (Table 1 ) were identified as showing a statistically significant (corrected p-value < 0.05) difference in methylation levels between SS cases and non-SS controls,

[0076] The inventors have also conducted genome-wide DNA methylation (DNAm) profiling using blood from individuals with Weaver syndrome (WVS), a disorder involving aberrant EZH2 function. Based on comparison of the DNA methylation profile from WVS individuals to those of non-WVS controls, the inventors have shown that DNA methylation profiles may be used in a test for early and accurate diagnosis of Weaver syndrome due to EZH2 pathogenic mutations. 55 CpG loci (Table 8) were identified as showing a difference in methylation levels between WVS cases and non-WVS controls.

I. Definitions

[0077] Terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art. [0078] As used herein, the term "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.

[0079] As used herein, "methylation" refers specifically to DNA methylation, 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-hydroxymethylcytosines (5-hmC).

[0080] As used herein, "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, genome build (NCBI hg 19, genome build 37), and +/- strand designation to unambiguously define each CpG locus. The genomic information is publically available through the UCSC genome browser at https://qenome.ucsc.edu/.

[0081 ] The term "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. For example, 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 "β value" or "beta value", which is calculated as:

β value = intensity of the methylated target (M)/(intensity of the unmethylated target (U) + intensity of the methylated target (M) + 100) A β value of zero indicates no methylation and a value of one indicates 100% methylation.

[0082] As used herein, the term "methylation status" refers to whether a specified target DNA site is methylated or not methylated. The target site may be an individual CpG locus, a region of DNA comprising multiple CpG loci, for example, a gene promoter or a gene. For example, a target site may have a methylation status of "methylated" or "hypermethylated" if the target has significantly higher methylation beta value in a SS (or WVS) specific control profile compared to a non-SS (or non-WVS) control profile. Conversely, a target site may have a methylation status of "not methylated" or "hypomethylated" if the target has significantly lower methylation beta value in a SS (or WVS) specific control profile compared to a non-SS (or non-WVS) control profile.

[0083] As used herein, the term "delta beta" or "delta β" refers to the difference between the p vafue of a methylation target in two different samples, for example, the β value of a methylation target in a SS (or WVS) specific control profile and the β value of the same methylation target in a non-SS (or non-WVS) control profile.

[0084] As used herein 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. As used herein, the term "enhancer" refers to a cis-acting region of DNA that is located up to 1 Mbp (upstream or downstream) of a gene.

[0085] As used herein, the term "sample methylation profile" or "sample 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 the methods disclosed herein is referred to as a sample profile.

[0086] In some embodiments, 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, or not have, Sotos syndrome or Weaver syndrome. In such cases, the historical methylation data can be a value that is continually updated as further samples are collected and individuals are identified as SS or not-SS or WVS or not-WVS. It will be understood that 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.

[0087] For example, a "SS specific control profile" or "SS 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 SS and an NSD1 pathogenic mutation. Similarly, a "non-SS 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 SS, In certain embodiments, 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.

[0088] Further, a "WVS specific control profile" or "WVS 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 WVS and an EZH2 pathogenic mutation. Similarly, a "non-WVS 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 WVS. In certain embodiments, 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

[0089] As used herein, 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.

[0090] As used herein, the term "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%.

[0091] As used herein, the term "specificity" refers the ability of a test to correctly identify those patients without the disease or disorder, such that a 100% specificity indicates a test that correctly identifies all patients without the disease or disorder. Specificity is calculated as:

Specificity = (True Negatives)/(True Negatives + False Positives). A high specificity as used herein refers to a specificity of greater than 50%.

[0092] As used herein, the term "CpG" or "CG" site refers to cytosine and guanosine residues located sequentially (5'->3') in a polynucleotide DNA sequence. The term "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. As used herein the term "CpG island shore" refers to a region of DNA occurring within 2kbp (upstream or downstream) of a CpG island. As used herein the term "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. As used herein the term "distance from TSS" refers to the genomic difference in base pairs between specific CpG locus and the nearest transcription start site.

[0093] As used herein, a first CpG locus is "associated" with a second CpG locus, if the methylation status at the first locus is reasonably predictive of the methyiation 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.

[0094] As used herein, 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.

[0095] The term "subject" as used herein refers to a human subject and includes, for example, a fetus.

[0096] The terms "complementary" or "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). As used herein, the term "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. Or, there may be "complete" or "total" complementarity between the polynucleotides. Those skilled in the art of 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.

[0097] The term "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.

[0098] The stringency may be selected based on the conditions used in the wash step. For example, 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. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C for 15 minutes.

[0099] By "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. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5°C - 16.6 (Log10 [Na+]) + 0.41 (%(G+C) - 600/I), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule 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 fina! wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In an embodiment, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's solution/1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC/0.1 % SDS at 60°C for 15 minutes. 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., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000, Third Edition.

[00100] The term "oligonucleotide" as used herein 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. The term "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.

[00101 ] As used herein, 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 (PGR), 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).

II. Methods

[00102] As set out in Table 1 , the instant disclosure identifies 7085 distinct CpG ioci (the "7K" CG sites), each of which show a statistically significant (corrected p-value < 0.05) difference in methylation levels between individuals with SS and non-SS controls over the tested population. As described in the Examples, the methylation levels of the disclosed loci, or a subset thereof, may be used in diagnostic testing for SS, with up to 100% sensitivity and specificity, ft will be understood that the sensitivity and specificity of the methods described will tend to increase with the number of CpG loci or sites selected for testing (i.e. the size of the signature), to a maximal sensitivity/specificity of 100%. However, signatures utilizing fewer CpG loci, for example as few as 7 (Example 4), are described herein which retain greater than 50% sensitivity and specificity and are usefui for assessing likelihood of Sotos syndrome.

[00103] As set out in Table 8, the instant disclosure also identifies 55 distinct CpG loci, each of which show a difference in methylation levels between individuals with WVS and non-WVS controls over the tested population. As described in the Examples, the methylation levels of the disclosed loci, or a subset thereof, may be used in diagnostic testing for WVS, with up to 100% sensitivity and specificity. It will be understood that the sensitivity and specificity of the methods described will tend to increase with the number of CpG loci or sites selected for testing (i.e. the size of the signature), to a maximal sensitivity/specificity of 100%. However, signatures utilizing fewer CpG loci, are described herein which retain greater than 50% sensitivity and specificity and are useful for assessing likelihood of Weaver syndrome.

[00104] Useful methylation signatures according to the described methods are not intended to be limited to the 7K CG sites of Table 1 and the 55 CG sites of Table 8, but are intended to include associated CpG loci, and associated gene and non-gene regions. DNA methylation at a single CpG locus can predict DNA methylation of multiple other loci residing in near genomic proximity or overlapping CpG islands. Accordingly, "associated" loci and regions are loci and regions, the methylation levels or status of which may be reasonably predicted by the methylation levels or status of one or more of the CpG loci of Table 1 or Table 8. 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.

[00105] Accordingly, an aspect of the disclosure provides a method of detecting and/or screening for Sotos syndrome (SS), or an increased likelihood of SS, in a human subject, comprising determining a sample methylation profile from a sample of DNA from said subject, said sample profile comprising the methylation level of at least one, optionally at least 7, at least 10, at least 17, at least 25, at least 41 , at least 57, at least 87, at least 124, at least 156, at least 220, at least 287, at least 399, at least 549, at least 726, or all CpG loci selected from (i) Table 1 and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i). [00106] Another aspect of the disclosure provides a method of detecting and/or screening for Weaver syndrome (WVS), or an increased likelihood of WVS, in a human subject, comprising determining a sample methylation profile from a sample of DNA from said subject, said sample profile comprising the methylation level least one, optionally at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or all CpG loci from (i) Table 8 and/or (ii) associated CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci of (i).

[00107] Methods of DNA methylation profiling of target genomic regions are generally known in the art (Stevens et al 2013, Harris et al 2010 and Hirst 2013).

[00108] For example, 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.

[00109] In an embodiment, 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 oligonucieotide-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. A non-limiting example of such an agent includes a "microarray", comprising an ordered set of probes fixed to a solid surface that permits analysis such as methylation analysis of a plurality of genomic targets sequences.

[00110] According to the methods described herein, similarity of the DNA methylation profile from a sample to one or more control profiles, may be used to identify individuals having Sotos syndrome, or an increased likelihood of having Sotos syndrome. For example, in an embodiment, 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 SS specific profile; (ii) a low level of similarity to a non-SS control profile; and/or (iii) a higher level of similarity to a SS specific profile than to a non-SS control profile indicates the presence of, or an increased likelihood of, SS.

[0011 1 ] Similarity of the DNA methylation profile from a sample to one or more control profiles, may be used to identify individuals having Weaver syndrome, or an increased likelihood of having Weaver syndrome. For example, in an embodiment, 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 profi!e to a WVS specific profile; (ii) a low level of similarity to a non-WVS control profile; and/or (iii) a higher level of similarity to a WVS specific profile than to a non-WVS control profiie indicates the presence of, or an increased likelihood of, WVS.

[00112] It will be appreciated that the 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, Sotos syndrome or Weaver syndrome. In such cases, the historical methylation data can be a value that is continually updated as further samples are collected and individuals are identified as SS or not-SS or WVS or not-WVS. For example, the control database may be stored on an online database, which is continually updated with methylation data from diagnosed SS and non-SS patients and/or diagnosed WVS and non-WVS patients. It will be understood that the control profile represents an average of the methylation levels for selected CpG loci as described herein.

[00113] In an embodiment, the "SS specific 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 SS. Similarly, in an embodiment, the "non-SS 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 SS. In certain embodiments, 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. In other embodiments, the sample profile and control profile are derived from different tissues. In certain other embodiments, the SS specific control profile and the non-SS control profile are derived from historical data and can indicate similarity of a sample to either the SS or non-SS profiles.

[00114] In another embodiment, the "WVS specific 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 WVS. Similarly, in an embodiment, the "non-WVS 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 WVS. In certain embodiments, 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. In other embodiments, the sample profile and control profile are derived from different tissues. In certain other embodiments, the WVS specific control profile and the non-VWS control profile are derived from historical data and can indicate similarity of a sample to either the VWS or non-VWS profiles.

[00115] 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.

[00116] For example, in an embodiment, similarity may be measured using hierarchical clustering, optionally using Manhattan distance. For example, unsupervised hierarchical clustering of the said sample with the SS specific control profile indicate similarity to an SS and if the said sample cluster with the non-SS control profile, this wiil indicate similarity to non-SS control profile.

[001 17] The Manhattan distance function computes the distance that would be traveled to get from one data point to the other if a grid-iike path is followed. The Manhattan distance between two items is the sum of the differences of their corresponding components.

[00118] The formula for this distance between a point X=(X1 , X2, etc.) and a point Y=(Y1 , Y2, etc.) is:

Where n is the number of variables, and Xi and Yi are the values of the variable, at points X and Y respectively.

[00119] In another embodiment, 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 . In an embodiment, the correlation coefficient may be a linear correlation coefficient, for example, a Pearson product-moment correlation coefficient.

[00120] A Pearson correlation coefficient (r) is calculated using the following formula:

[00121 ] In one embodiment, x and y are the beta values for various CpG loci in a sample profile and a control profile, respectively.

[00122] In an embodiment, 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.

[00123] It will be appreciated that any "correlation value" which provides a quantitative scaling measure of similarity between methylation profiles may be used to measure similarity.

[00124] A sample profile may be identified as belonging to an individual with SS, or an increased likelihood of SS, where the sample profile has high similarity to the SS profile, low similarity to the non-SS profile, or higher similarity to the SS profile than to the non-SS profile. Conversely, a sample profile may be identified as belonging to an individual without SS, or an decreased likelihood of SS, where the sample profile has high similarity to the non-SS profile, low similarity to the SS profile, or higher similarity to the non- SS profile than to the SS profile. [00125] For example, in an embodiment, a sample profile may be identified as belonging to an individual with SS, or an increased likelihood of SS, based on calculation of a Sotos Syndrome Score, which generally is defined by the following formula:

SS score(B) = r (B, SS profile) - r (B, control profile)

where r is the Pearson correlation coefficient, and B is a vector of DNA methylation levels across the selected CpG loci.

[00126] A sample profile with a positive Sotos Syndrome Score is more similar to the SS specific profile across the selected CpG loci, and is therefore classified as "SS"; whereas a sample with a negative Sotos Syndrome Score is more similar to the non-SS profile across the selected CpG loci, and is classified as "not SS".

[00127] A sample profile may be identified as belonging to an individual with WVS, or an increased likelihood of WVS, where the sample profile has high similarity to the WVS profile, low similarity to the non-WVS profile, or higher similarity to the WVS profile than to the non-WVS profile. Conversely, a sample profile may be identified as belonging to an individual without WVS, or an decreased likelihood of WVS, where the sample profile has high similarity to the non-WVS profile, low similarity to the WVS profile, or higher similarity to the non-WVS profile than to the WVS profile.

[00128] For example, in an embodiment, a sample profile may be identified as belonging to an individual with WVS, or an increased likelihood of WVS, based on calculation of a Weaver Syndrome Score, which generally is defined by the following formula:

WVS score (B) = r (B, WVS profile) - r (B, control profile)

where r is the Pearson correlation coefficient, and B is a vector of DNA methylation levels across the selected CpG loci. [00129] A sample profile with a positive Weaver Syndrome Score is more similar to the WVS specific profile across the selected CpG loci, and is therefore classified as "WVS"; whereas a sample with a negative Weaver Syndrome Score is more similar to the non-WVS profile across the selected CpG loci, and is classified as "not WVS".

[00130] As used herein the term "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.

[00131 ] Median methylation levels for SS and non-SS cases reported in Table 1 were identified using whole blood samples. However, the distinct methylation profiles observed in SS cases was also present in fibroblasts and amniocytes. For example, as shown in Example 3, a large subset of the CpG sites from the SS specific DNAm signature shared similar DNA methylation profiles in both blood and fibroblast tissues suggesting that the SS-specific profiles will be present in multiple tissues of individuals with Sotos syndrome and NSD1 mutations.

[00132] Another aspect provides, a method of detecting and/or screening for Sotos syndrome (SS), or an increased likelihood of SS, in a human subject, comprising determining the methylation level of at least one, optionally at least 6, at least 12, at least 17, at [east 18, at least 24, at least 33, at least 49, at least 67, at least 72, at least 101 , at least 127, at least 164, at least 212, at least 275, or all the genes from Table 1 in a sample comprising genomic DNA from the subject, wherein a difference in methylation levels of genes selected from Table 1 , relative to a non-SS control profile is indicative of SS, or an increased likelihood of SS, in the subject.

[00133] Another aspect provides, a method of detecting and/or screening for Weaver syndrome (WVS), or an increased likelihood of WVS, in a human subject, comprising determining the methylation level of at least one, optionally at least 2, at least 4, at least 6, or all the genes from Table 8 in a sample comprising genomic DNA from the subject, wherein a difference in methylation levels of genes selected from Table 8, relative to a non-WVS control profile is indicative of WVS, or an increased likelihood of WVS, in the subject.

[00134] Diagnostic certainty for Sotos syndrome aids in medical management by enabling targeted medical testing and intervention. Targeted testing includes, but is not limited to, echocardiogram to screen for congenital heart defects, renal ultrasound to screen for congenital renal anomalies, audiology and ophthalmology assessment to screen for hearing and vision problems and active clinical monitoring to screen for scoliosis. In light of the increased incidence of non-febrile seizures, thorough electrophysiologic investigation of any suspected seizure activity would be indicated. Knowledge of the increased risk for tumors directs more aggressive investigation of common symptoms to enable early diagnosis and to maximize the opportunity for early intervention. In addition, neuropsychological assessment to screen for developmental/learning disabilities (individuals with Sotos syndrome have mild to moderate intellectual disability) and behavioural problems (e.g. aggression) provides the opportunity for early identification and optimal intervention. There is also an increased risk for other neuropsychological issues including ADHD and ASD, for which early diagnosis provides the opportunity for early intervention and improved outcomes. In summary, early identification of the above medical and cognitive issues provides the opportunity for an enhanced quality of life for individuals with Sotos syndrome.

[00135] Molecular confirmation of a diagnosis of Weaver syndrome is of value as it aids in medical management by enabling targeted medical testing and intervention and eliminating the need for screening for symptoms of other overgrowth syndromes. Targeted intervention for patients with Weaver syndrome includes, but is not limited to, active ciinical monitoring to screen for scoliosis and follow-up of camptodactyly to monitor the need for surgical intervention. In addition, knowledge of the increased risk for tumors directs more aggressive investigation of common symptoms to enable early diagnosis and to maximize the opportunity for early intervention. In addition, neuropsychological assessment to screen for developmental/learning disabilities (individuals with Weaver syndrome typically have an intellectual disability) provides the opportunity for early identification and optimal intervention. Individuals with Weaver syndrome also present with hypotonia and ligamentous laxity and may benefit from physiotherapy. In summary, early identification of the above medical and cognitive issues provides the opportunity for an enhanced quality of life for individuals with Weaver syndrome.

[00136] Accordingly, an aspect provides a method of assigning a course of management for an individual with Sotos syndrome (SS), or an increased likelihood of SS, comprising:

a) identifying an individual with SS or an increased likelihood of SS, according the methods described herein; and

b) assigning a course of management for SS and/or symptoms of a SS, comprising i) testing for at least one medical condition associated with SS and ii) applying an appropriate medical intervention based on the results of the testing.

[00137] In one embodiment, the medical condition is selected from congenital heart defects, congenital renal abnormalities, hearing problems, vision problems, scoliosis, seizure activity, developmental/learning disabilities, behavioural problems and neuropsychological issues.

[00138] Another aspect provides a method of assigning a course of management for an individual with Weaver syndrome (WVS), or an increased likelihood of WVS, comprising:

a) identifying an ^' individual with WVS or an increased likelihood of WVS, according the methods described herein; and

b) assigning a course of management for WVS and/or symptoms of a WVS, comprising i) testing for at least one medical condition associated with WVS and ii) applying an appropriate medical intervention based on the results of the testing.

[00139] In one embodiment, the medical condition is selected from scoliosis, camptodactyly, tumor growth, developmental/learning disabilities, intellectual disabilities, hypotonia and ligamentous laxity.

[00140] As used herein, the term "a course of management" refers to any testing, treatment, medical intervention and/or therapy applied to an individual with SS or WVS and/or symptoms of SS or WVS. Medical interventions include, but are not limited to, pharmaceutical treatments, surgical procedures, utilization of medical devices such as hearing aids or glasses, physical or occupational therapy and behavioral or cognitive therapy.

III. Kits

[00141] Another aspect provides a kit for detecting and/or screening for Sotos syndrome, or an increased likelihood of SS, in a sample, comprising:

a) at least one detection agent for determining the methy!ation level of:

i) at least one, optionally at least 7, at least 10, at least 17, at least 25, at least 41 , at least 57, at least 87, at least 124, at least 156, at least 220, at least 287, at least 399, at least 549, at least 726, or all CpG loci selected from

Table 1 or CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci in Table 1 , and/or

ii) at least one, optionally at least 6, at least 12, at least 17, at least 18, at least 24, at least 33, at least 49, at least

67, at least 72, at least 101 , at least 127, at least 164, at least 212, at least 275, or all the genes from Table 1 ; and b) instructions for use. [00142] Another aspect provides a kit for detecting and/or screening for Weaver syndrome, or an increased likelihood of WVS, in a sample, comprising:

a) at least one detection agent for determining the methyiation level of:

i) at least one, optionally at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or aii CpG loci from Table 8 or CpG loci residing within 300 nucleotides, optionally within 150 nucleotides, of the CpG loci in Table 8, and/or

ii) at least one, optionally at least 2, at least 4, at least 6, or all the genes from Table 8; and

b) instructions for use.

[00143] In an embodiment, the kit further comprises bisulfite conversion reagents, methylation-dependent restriction enzymes, methylation-sensitive restriction enzymes, PCR reagents, probes and/or primers.

[00144] In an embodiment, the kit further comprises a computer- readable medium that causes a computer to compare methyiation levels from a sample at the selected CpG loci or genes to one or more control profiles and compute a correlation value between the sample and control profile.

[00145] Other features and advantages of the disclosure will become apparent from the following detailed description. It should be understood, however, that the description and the specific examples while indicating preferred embodiments are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this description of various embodiments.

[00146] The following non-limiting examples are illustrative of the present disclosure: EXAMPLES

Example 1 : Identification of DNAm signature in SS patients with NSD1 pathogenic mutations

Materials and Methods

Sample Collection

Discovery cohort

[00147] Individuals with a clinical diagnosis of Sotos syndrome and a pathogenic NSD1 mutation were recruited through an REB approved research study at the Hospital for Sick Children in Toronto, Ontario. A clinical diagnosis of Sotos syndrome was established based on the following criteria: height +/- weight greater than 2 SD above the mean, macrocephaly (OFC >2SD), developmental delay and characteristic facial gestalt. Informed consent was obtained from parents of ali participants and assent was obtained from participants, as appropriate based on age. DNA from biood samples was extracted by standard methods. In ali, 19 individuals, including one familial case with three affected individuals (father and two children), were included. All of these patients had a pathogenic NSD1 mutation, including a whole gene deletion or truncating mutation. In addition, six individuals with an overgrowth phenotype (some with a suspected clinical diagnosis of Sotos syndrome and some without the Sotos' gestalt) who were identified to have a missense mutation (single nucleotide substitution) that was classified as a variant of unknown significance, were also enrolled in the study. All patients with missense mutations were examined in person, or via medical records and photographs, independently by two reviewers with extensive clinical experience with Sotos syndrome (RW), who were blinded to the methylation results. Patients were classified into one of three phenotypic categories: (1) "typical Sotos syndrome", (2) "possible Sotos syndrome", and (3) "unlikely Sotos syndrome". There was 100% concordance (6/6) between the clinical classification of both reviewers and the methylation status. Specifically, four patients were categorized as "typical Sotos syndrome", all of whom had the DNAm signature and two patients were categorized as "unlikely Sotos syndrome", neither of whom had the signature. The specific NSD1 mutations and clinical features of the patient cohort are summarized in Table 2.

Validation cohort

[00148] An additional 19 patients with a clinical diagnosis of Sotos syndrome and confirmed pathogenic NSD1 mutation (whole gene deletion or truncating mutations) and 10 patients with missense mutations were obtained from the University of Hong Kong. All patients with missense mutations for whom photographs and medical records were available (6/10) were assessed independently by two experienced clinicians who were blinded to the methylation results. Patients were assigned to one of the three phenotypic categories stipulated above. The clinicians' assessments were concordant for 5/6 patients. Specifically, two of the patients were categorized by both clinicians as "unlikely Sotos"; these patients did not have the methylation signature. Two of the patients were categorized by both clinicians as "typical Sotos"; these patients did have the methylation signature. One patient with the methylation signature was categorized by both clinicians as "possible Sotos". In a single case, the clinicians' opinions were discordant, with one categorizing the patient as "possible Sotos syndrome" and the other as "unlikely Sotos syndrome". Of note, this patient had the most limited clinical information available for review.

Control cohort

[00149] Genomic DNA from 53 consented control blood samples recruited at The Hospital for Sick Children following was used. DNA methylation data from an additional 1056 control blood samples were downloaded from the gene expression omnibus (GEO) public database fhttp://www. ncbi.nlm.nih.gov/geo/). This database is a repository of high throughput gene expression data and hybridization arrays, chips and microarrays. DNA methylation analysis

[00150] 1 microgram of genomic DNA was subjected to bisulfite conversion using the Qiagen EZ DNA Methylation™ kit (Qiagen), according to the manufacturer's protocols. Modified genomic DNA was processed as described in the Infinium® Assay Methylation Protocol Guide Rev. C (November 2010) and analyzed on Infinium® HumanMethylation450 BeadChips (lllumina). Probes that had missing values, had detection p-value <0.01 and non-specific probes as well as known bound targets with known sequence variants [Chen et al, 2013a] were removed. The final set contained 424,586 probes.

Differential DNA methylation analysis

[00151 ] To identify the differentially methylated CpG sites, the DNAm distributions in Sotos cases were compared against controls at each CpG site. To account for the influence of the family relationships among three of the Sotos subjects, three separate testing trials were formed, each time combining 16 non-familial Sotos cases with only one family member. The resulting set of 17 Sotos subjects was compared to 53 controls for each of the 424,586 available CpG sites, using a non-parametric Mann-Whitney U test. A stringent Bonferroni correction for multiple testing was applied to the results in each trial. In order to ensure robust results, only the CpG sites that were significant at the confidence level a=0.05 in ail three trials were retained, i.e. with any choice of the family representative among the Sotos subjects. As many as 28458 CpG sites satisfied this criterion. Finally, an additional effect- size criterion requiring at least 20% difference in DNA methylation between the Sotos and the control groups in each of the three trials was applied. This filter reduced the NSD1 signature to the robust set of 7085 CpG sites, which were selected for further characterization, and for specificity and sensitivity testing in independent control and SS case cohorts. Sotos Syndrome Score and Classification Model

[00152] A simple classification model based on the NSD1 ^+/- DNAm signature in blood was developed. At each of the 7K CGs, a median DNAm level was computed across all 19 SS subjects in the original Discovery cohort. This resulted in a reference profile of the NSD1 ^+/- Sotos DNAm level over the 7K CGs, which was robust to outliers. Similarly, a robust median-DNAm reference profile for the 53 healthy control subjects was created. The classification of each new DNAm sample was based on extracting a vector B _7K of its DNAm values in the 7K CGs, and comparing B _7K to the two reference profiles computed above. A Sotos Syndrome Score was defined as:

SS score(B) = r (B _7Kl SS profile) - r (B _7K , control profile)

where r is the Pearson correlation coefficient. A simple classification model was developed based on scoring each new DNAm sample using SSS: asample with a positive Sotos Syndrome Score is more similar to the SS reference profile based on the 7K CGs, and is therefore classified as "SS"; whereas a sample with a negative Sotos Syndrome Score is more similar to the normal reference profile, and is classified as "not SS".

Results

[00153] DNA methylation was compared in peripheral blood from SS cases (NSD1 ^+/- ; n=19) to controls (n=53), Initially known pathogenic mutations including NSD1 whole gene deletions and truncating mutations {NSD1*' ^~ ) were used (Table 2) as well as one familial case of Sotos syndrome.

[00154] Genomic DNA obtained from whole blood was bisulfite treated and assessed using the lllumina Human Infinium® Methyiation450 BeadChip. After filtering for non-specific probes as previously described [Chen et al, 2013b], methylation was quantified (beta values; range: 0-1 ) at 424,586 CpG sites. The significance of differential DNA methylation between the Sotos and control samples was assessed at each CpG using a non-parametric Mann- Whitney U test with a stringent Bonferroni correction for multiple testing. A largely specific signature for NSD1 loss of function mutation was identified by selecting the set of probes that achieved statistical significance (corrected p<0.05) and had at least 20% difference in DNA methyiation levels between the Sotos and non-Sotos control groups (Figure 1 ). 7085 CpG sites (or 7K CGs) that were distributed across the genome were identified; 7038 CpG sites (99.3%) demonstrated loss of DNA methyiation (Table 1 ) whereas only 47 CpG sites (0.6%) exhibited gain of methyiation in SS compared to controls. Using unsupervised hierarchical clustering of the 7K CGs, all samples with NSD1 ^+/- faithfully clustered as a distinct group separate from controls (Figure 1 ).

[00155] The NSD1 ^+/- DNAm signature was used to develop a predictive model, which classifies each new sample based on its DNAm profile as either SS or not-SS, using the Sotos Syndrome score (SS score; see above). The specificity of the SS score was tested using a iarge independent test set of normal DNAm blood samples (n=1056 subjects) extracted from the GEO database (www.ncbi.nlm.nih.gov/geo/). Each of the 1056 samples from the GEO coilection received a negative SS score and were classified as "not SS" (Figure 2), suggesting a 100% specificity of the classification model. The size and the diversity of data sources in the GEO test dataset suggest that new samples from norma! patients are highly likely to be classified correctly as "not Sotos" by using the NSD1 ^+/- DNAm signature.

[00156] The sensitivity of the SS score was also tested as a predictor of SS using a replication cohort of cases with whole gene deletions and truncating mutations (n=19) from Hong Kong (Table 3).

[00157] The sensitivity was 100%, as each of the SS subjects in the replication cohort received a positive SS score (Figure 3).

[00158] To further assess the value of the highly specific NSD1 ^+/- DNA methyiation signature, the SS DNA methyiation profile with the DNA methyiation profile of eight subjects with a clinical diagnosis of Weaver syndrome (OMIM 277590) who had confirmed mutations in a different histone ethyltransferase (Table 4), Enhancer of Zeste, Drosophila, Homolog 2 or EZH2 [Gibson et ai, 2012; Tatton-Brown et al, 2011].

[00159] All Weaver syndrome patients with EZH2 ^+/- mtations received a strongly negative SS score (between -0.151 and -0.105) and hence were classified confidently as "not Sotos". Furthermore, hierarchical clustering grouped the Weaver syndrome cases together with the controls, while the SS individuals maintained their distinct cluster. Clearly, the results indicate that the 7K CGs allow the molecular distinction of two clinically overlapping overgrowth syndromes and further demonstrates the specificity of the NSD1 ^+/- DNAm signature for SS (Figure 3).

Example 2: DNAm signature is capable of distinguishing between pathogenic variants in N$D1 and benign variants in NSD1

[00160] The efficiency of the NSD 1 ^+/- DNAm signature to functionally classify NSD1 variants of unknown significance (VUS) as pathogenic or benign was investigated. DNA samples from 16 individuals with missense mutations in NSD1 were blinded in terms of inheritance and associated clinical features. As shown in Figure 3, using average Beta DNA methylation levels at the 7K CGs, a correlation matrix was generated with unsupervised clustering in which nine of 16 samples clustered as a separate group away from the other 7 samples. Both the SS score-based classification (Figure 3) and the unsupervised hierarchical clustering confirmed that the group of 9 subjects (Group I) carry missense mutations that can clearly be classified as pathogenic since all of them received positive SS score and also clustered with SS subjects. The remaining 7 subjects received negative SS score and clustered with controls indicating benign variant. The classification of variants into benign and pathogenic was compared to 2 other approaches. First, two highly experienced clinical geneticists reviewed the available photos and clinical information for these cases without knowledge of the molecular data for individual cases. Then the predicted pathogenicity of the missense mutations in the cohort utilizing the NSD1 ^+/- DNAm signature was compared to several different prediction algorithms (Table 2) revealing that the DNAm at the NSD1 ^+/- was the most congruent with the clinical diagnosis established by highly experienced clinicians.

Example 3: NSD1 ^+/- methyiation signature is evident across multiple cell lineages and the genes associated with the signature are enriched in genes involving neural and cellular development pathways.

[00161] It was hypothesized that NSD1 mutations impact the epigenome of multiple cell lineages by generating specific DNA methyiation tags at specific genomic regions during embryonic development. Therefore, fibroblast-derived DNA samples from 3 SS subjects with truncating mutations in NSD1 were tested and compared to 4 control fibroblast samples. Using unsupervised hierarchical clustering of the NSD1 ^+/- 7K CGs, it was found that the 3 SS fibroblast clustered separately from control fibroblasts (Figure 4). Comparing these data to the SS DNAm profile in blood, it was found that a large subset of the CpG sites from the SS specific DNAm signature shared similar DNA methyiation profiles in both blood and fibroblast tissues.

[00162] Finally, the DNAm signature was investigated for its potential to elucidate the molecular pathophysiology of SS. Analysis of the genomic locations of the 7K CpGs in the NSD1 ^+/- DNA methyiation signature showed that the majority of these CpGs mapped to enhancers and CpG island shores. Enrichment analyses for biological and molecular functions using DAVID [Huang da et al, 2009] identified enrichment in neural and cellular development pathways (Benjamini-Hochberg corrected p<0.05) highlighting the main clinical features associated with SS (i.e. overgrowth and developmental delay).

Example 4. Sensitivity and Specificity of the NSD1+I- Signature at Various Delta-Beta Cutoffs.

[00163] A series of leave-one-out (LOO) cross-validations were performed on the combined dataset (composed of 53 not SS and 36 SS). At each LOO iteration, one sample was removed from the dataset for the subsequent validation step. The remaining samples were used to generate median DNAm profiles for the SS group and for the non-SS group, respectively. Thereafter the previously retained validation sample was classified in terms of its Pearson correlation to either the SS profile or the non- SS profile, using only the significant CpGs as determined by the p-value and delta-beta thresholds. This classification was compared to the sample's true status (SS or non-SS). Based on these LOO results over all 36 (SS) + 53 (non-SS) samples, the specificity and sensitivity were estimated for various threshold combinations (Table 5 and Figure 5). Table 5 includes Recall, Precision and F-measure values. Recall is similar to sensitivity. Precision is calculated as true positives over the sum of true and false positives. F- measure combines precision and recall and is an alternative way to measure sensitivity and specificity.

Example 5. DNA methylation analysis of Weaver syndrome samples

[00164] 52 control samples and 7 Weaver samples with EZH2+/- variants (Table 6) were used for differential DNA methylation analysis. Three EZH2 variants are described in Table 7. After filtering for non-specific probes as per Chen et al, 2012, 424586 out of 485500 CpG sites were used for the differential DNA methylation analysis. Statistical analysis was performed using Qlucore Omics Explorer. Two group comparisons were performed using two- tailed tests and accounting for gender to assess for differential methylation. At a q<0.05 (Benjamini correction), 3126 CpG sites were identified to be highly significant. The q-value represents the corrected p-value, using the Benjamini Hochberg method to account for multiple testing. Figure 6 shows unsupervised hierarchical clustering of the EZH2+/- DNA methylation signature.

[00165] A subset of 6 subjects with EZH2+/- clustered together separate of all control samples and one subject with EZH2+/- (Subject F0123) clustered with the control samples. Figure 7 shows a principal component analysis revealing a clear separation between EZH2+/- and the controls. Principal Component Analysis (PCA) is a well-established mathematical technique for reducing the dimensionality of data, while keeping as much variation as possible. PCA achieves dimension reduction by creating new, artificial variables called principal components. Each principal component is a linear combination of the observed variables. PCA is an unsupervised method, meaning that no information about groups is used in the dimension reduction. Accordingly, PCA shows a visual representation of the dominant patterns in a data set. By calculating, for example, the first three principal components, and visualizing the samples in this three-dimensional space, a visualization is created containing more of the variance in the original data than any other trio of linear combinations, to provide a three-dimensional sample representation.

[00166] Subsequent Insilco analysis using the CltnVar database identified that the variant in F0123 is likely a benign variant. Thus, this data highlights the validity of the EZH2+/- DNA methylation signature for determining the functionality of single nucleotide EZH2 variants,

[00167] To identify a minimal signature that can classify Weaver subjects separate from control samples, a combination of different fold change threshold and hierarchical clustering was used to select the most relevant CpG sites that will maintain the separation. At a fold change of 1.12 in methylation levels in addition to q<0.05 (Benjamini correction), there are a total of 55 CpG sites contained within 8 genes (Figures 8 and 9 and Table 8). A fold change of at least 1.12 at q<0.05 (Benjamini correction) maintains the separation between Weaver samples and control samples when analyzed by hierarchical clustering. In Table 8, both the "difference" and "fold change" are provided for each CpG locus. The "difference" is the "average difference" and represents the difference between the means of methylation level between the Weaver samples and the control samples. The "fold change" is the average difference to the power of 2. [00168] 40 of the CpG sites in Table 8 correspond to one gene cluster on chromosome 7 (the HOXA gene cluster) as shown in Figure 10.

Example 6. Sensitivity and Specificity of the EZH2 Signature at Various Delta-Beta Cutoffs.

[00169] A series of leave-one-out (LOO) cross-validations are performed on the Weaver dataset. At each LOO iteration, one sample is removed from the dataset for the subsequent validation step. The remaining samples are used to generate median DNAm profiles for the WVS group and for the non- WVS group, respectively. Thereafter the previously retained validation sample is classified in terms of its Pearson correlation to either the WVS profile or the non-WVS profile, using only the significant CpGs as determined by the p- value and delta-beta thresholds. This classification is compared to the sample's true status (WVS or non-WVS). Based on these LOO results, the specificity and sensitivity is estimated for various threshold combinations.

[00170] While the present disclosure has been described with reference to what are presently considered to be the examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00171] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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