DNA Methylation Episignatures in Neurodevelopmental Disorders Associated with Large Structural Copy Number Variants: Clinical Implications

Large structural chromosomal deletions and duplications, referred to as copy number variants (CNVs), play a role in the pathogenesis of neurodevelopmental disorders (NDDs) through effects on gene dosage. This review focuses on our current understanding of genomic disorders that arise from large structural chromosome rearrangements in patients with NDDs, as well as difficulties in overlap of clinical presentation and molecular diagnosis. We discuss the implications of epigenetics, specifically DNA methylation (DNAm), in NDDs and genomic disorders, and consider the implications and clinical impact of copy number and genomic DNAm testing in patients with suspected genetic NDDs. We summarize evidence of global methylation episignatures in CNV-associated disorders that can be used in the diagnostic pathway and may provide insights into the molecular pathogenesis of genomic disorders. Finally, we discuss the potential for combining CNV and DNAm assessment into a single diagnostic assay.


Introduction
Neurodevelopmental disorders (NDDs) are a class of neurological and neuropsychiatric conditions that manifest in childhood during the developmental phase and persist throughout life [1]. They affect development of the central nervous system and can lead to brain dysfunction, resulting in limitations or impairment in cognition, motor performance, vision, hearing, speech and behavior [2]. NDDs include, but are not limited to, autistic spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD) and epilepsy, all of which show high rates of comorbidity and phenotypic overlap [3]. They are estimated to affect approximately 3% of children worldwide [4,5] and therefore, collectively represent a significant impact to families and health care systems.
NDDs present with a broad range of genetic and phenotypic heterogeneity, and clinical presentations are often non-specific. Genetics plays an important role in the etiology of hereditary NDDs. Genetic mutations associated with NDDs vary in size from single nucleotide variants (SNVs) to whole chromosome aneuploidies [6]. Due to the phenotypic overlap exhibited, in addition to targeted gene sequencing approaches, genetic testing often involves global genomic screening including chromosomal microarray analysis (CMA), exome or whole genome sequencing (WES, WGS), or classically G-banded chromosome karyotyping (karyotyping). CMA, considered the first-tier diagnostic test for patients with NDDs, has been used clinically for nearly two decades [7,8] to detect structural imbalances involving deletion or duplication of genetic material, collectively termed copy number variants (CNVs). Whilst some of the first CMAs included bacterial artificial chromosome Considerable research to date has focused on genes within the CNV regions of genomic disorders and how dosage sensitivity may be responsible for the observed phenotypes. While this research has identified some causative or candidate genes for specific Mendelian disorders and phenotypes, the genetic contribution for the majority of the observed clinical phenotypes in CNV disorders are not well defined [21]. One example of a disorder where a contributory gene has been identified is the 5q35 deletion. 5q35 deletion is predominantly mediated by NAHR and is associated with Sotos syndrome 1, where haploinsufficiency of the nuclear receptor-binding set domain protein (NSD1) gene contained within this region is shown, on its own, to be causative for Sotos [27]. This is similar to findings in Smith-Magenis syndrome, where deletions of 17p11.2 are responsible for 90% of causative variants, while approximately 5% are the result of point mutations in the retinoic acidinduced 1 (RAI1) gene contained within this region [22][23][24][25][26][27][28]. This is in contrast to disorders such as 16p11.2 and 22q11.2 deletion and duplication syndromes where no single candidate gene has been identified.
Overall, CNVs may contribute to the clinical features observed in genomic disorders through dosage sensitivity, via haploinsufficiency (as described for Sotos and Smith-Magenis), triplosensitivity (e.g., 22q11.2 duplication syndrome) or imprinting effects (Prader-Willi and Angelman syndromes), or through disruption of gene expression via positional effects, including disruption of transcriptional regulatory elements and changes in the chromatin structure.
This 'diagnostic odyssey', the time from initial consultation to diagnosis, often involves multiple clinical evaluations and laboratory tests spanning years [35,36], resulting in significant social and economic burden on both families and health care systems. In addition to CMA as the first-tier screen [7], in males with DD it is often accompanied by assessment for Fragile-X syndrome (FRX). FRX is an X-linked dominant condition and the most common inherited cause of ID. FRX results from abnormal expansion of the CGG trinucleotide repeat (>200 repeats) located in the promoter of the fragile X messenger ribonucleoprotein 1 (FMR1) gene, resulting in promotor DNA hypermethylation and gene silencing [37]. FRX can also be the result of deletions of Xq27.3 containing the FMR1 gene [38]. Reflexive genetic testing, whereby the results of previous tests are used to guide further investigations, include DNA methylation (DNAm) analysis in individuals with CNVs at common imprinting loci, e.g., 15q11.1 and 11p15.5 regions. The average time to diagnosis in patients referred for genetic testing is estimated at 1-8 years [7,[35][36][37][38][39], and the cost to healthcare is often difficult to estimate or missing from research. Recent studies describe the substantial positive medical and psychosocial outcome of receiving a genetic diagnosis [40,41]. Therefore, development of novel diagnostic technologies or testing strategies to shorten the diagnostic odyssey or increase the diagnostic yield represent an ongoing priority in NDD research [42].
Many recent studies have demonstrated disruption of genomic DNAm as a functional consequence of genetic defects in patients with NDDs [43][44][45][46]. There is emerging evidence of similar DNAm disruptions as epigenetic biomarkers for CNV disorders and their associated clinical phenotypes.

The Role of Epigenetics in NDDs and Subsequent Episignature Mapping
Epigenetics refers to mitotically heritable gene regulatory mechanisms without changes in the DNA sequence [47,48]. Epigenetic regulation of gene expression occurs at the level of chromatin and typically involves processes that modify chromatin or histones, the proteins around which DNA is wrapped, or covalent modifications in the associated DNA molecule [49]. DNAm is the most extensively studied epigenetic modification and refers to the mechanism of addition or removal of a methyl group to cytosine nucleotides [50]. Most cytosines subject to DNAm are adjacent to guanine residues and referred to as CpG dinucleotides (CpGs) [50]. High density clusters of CpGs, often associated with gene promoters, are referred to as CpG islands [50]. Unmethylated (hypomethylated) CpGs and CpG islands are generally associated with open, transcriptionally accessible chromatin, while DNA hypermethylation correlates with compact, transcriptionally repressive chromatin [51]. The majority of CpGs in the human genome are methylated except for those contained within CpG islands [52]. Therefore, in addition to affecting chromatin states and stability, disruptions in DNAm patterns can alter gene expression [51].
An increasing number of chromatin and epigenetic regulatory genes are becoming implicated in a variety of NDDs. Mutations in these genes result in DNAm episignatures, whole genome methylation changes, which are routinely detectable in the peripheral blood of patients affected by these disorders [43]. An episignature is defined as a recurring epigenetic pattern associated with a common genetic or environmental etiology in a disorderspecific patient population. Episignatures are highly sensitive and specific biomarkers that can be used to help resolve ambiguous clinical and genetic findings, and for screening patients with suspected genetic conditions [45]. Episignatures have the potential to provide insight into functional effects of certain mutations and genomic alterations on widespread DNAm and their contribution to the pathophysiology of genetic disorders [46].
Histone modifications refer to the chemical modification of histone tails by processes including methylation, acetylation, phosphorylation and ubiquitination. Histone tails are loosely structured protein segments that can mediate interaction between nucleosomes, and their modifications can result in either condensed or more relaxed chromatin, ultimately exhibiting an effect on gene transcription, as well as accessibility of DNA to other chromatin remodeling factors, including those involved in DNA methylation. Our group and others have identified unique episignatures in multiple NDDs that are the consequence of mutations in genes associated with histone modification [44], including, e.g., Kabuki syndrome caused by mutations in the lysine-specific methyltransferase 2D gene (KMT2D) [53]. We have mapped episignatures in several other histone modifying genes including lysine-specific methyltransferase 2B (KMT2B), set domain-containing protein 2 (SETD2), creb-binding protein (CREBBP), lysine acetyltransferase 6A (KAT6A) and lysine demethylase 4B (KDM4B) [43]. In addition, unique episignatures have also been reported in genes associated with the removal of histone methylation marks, the so-called "eraser" genes, such as the histone lysine demethylase 5C gene (KDM5C) in Claes-Jensen syndrome [54]. Histone modifications work in concert with DNAm to affect chromatin remodeling and gene expression.
The DNAm reaction is catalyzed by enzymes known as DNA methyltransferases (DNMT), which are responsible for mediating the transfer of the methyl group from S-adenosylmethionine (SAM) to cytosine residues. Robust episignatures have been reported in NDDs caused by mutations in the DNA methyltransferase genes DNMT1, DNMT3A and DNMT3B [44,55]. These genes are involved in the establishment and maintenance of DNAm during DNA replication and are termed "writers" since they are responsible for the addition of the methyl group to cytosines. Unique episignatures in two disorders are associated with mutations in DNMT1, hereditary sensory neuropathy with dementia and hearing loss (HSNDHL), and autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCADN) ( [56,57]), while loss of function mutations in DNMT3A result in an episignature in Tatton-Brown-Rahman syndrome (TBRS) [44,58]. Mutations in DNMT3B, which cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome, also result in genomic defects in DNAm [59]. We recently demonstrated a genome-wide DNA hypermethylation episignature in a DNA demethylation gene Tet methylcytosine dioxygenase 3 (TET3), an "eraser" gene that opposes the writer function of DNMT1 [60]. Mutations in the highly conserved catalytic domain of TET3 cause Beck-Fahrner syndrome (BEFAHRS). Inheritance patterns of BEFAHRS vary and include autosomal dominant or recessive forms [61]. Through episignature mapping, we were able to differentiate between affected individuals with mono-and bi-allelic mutations [60].
To date, chromatin remodeling genes comprise the largest group of epigenetic modifier genes with mapped episignatures, e.g., truncating mutations in the SNF2-related CBP activator protein gene (SRCAP) result in an episignature specific for Floating-Harbor syndrome [62]. Schenkel et al. reported a unique methylation profile associated with mutations in the ATRX chromatin remodeler gene ATRX in alpha-thalassemia X-linked intellectual disability syndrome [63]. In addition, our group previously described a shared DNAm episignature in Coffrin-Siris and Nicolaides-Baraitser syndromes (NCBRS) [64], which are two phenotypically similar NDDs associated with mutations in subunits of the BAF chromatin remodeling complex (commonly referred to as BAFopathies). This study described a shared BAFopathies episignature and supported the findings from previous studies suggesting that these conditions represent a disease spectrum rather than two distinct disorders [65]. Furthermore, this study indicates that methylation analysis may uncover or provide further support for the "relatedness" of genes and their disorders. In a subsequent study by our group, we described a new syndrome involving the BAF complex and the SWI/SNF-related matrix-associated, actin-dependent regulator of the chromatin gene (SMARCA2)-a gene reported in multiple individuals with NCBRS. This new syndrome was identified based on unique methylation patterns observed in individuals with intragenic variants located in the helicase domain of the SMARCA2 gene compared to individuals with pathogenic variants located outside the helicase domain [66]. In support of these findings, clinical features of patients with SMARCA2 helicase domain mutations exhibited a common phenotype distinct from NCBRS. Similarly, functional studies in yeast supported a different molecular mechanism underlying these two disorders [66]. Therefore, by analyzing variants from multiple regions within a gene, we were able to identify two unique episignatures and uncover functional data to explain the phenotypic differences seen between patients harboring variants in the same gene, resulting in the discovery of a new syndrome.
Interestingly, two distinct domain-specific episignatures have also been described in Helsmoortel-van der Aa syndrome associated with dominant negative truncating mutations in the activity-dependent neuroprotector homeobox gene (ADNP), which has chromatin regulatory functions [67]. These signatures were partially opposing, with mutations in the N-and C-terminus resulting in a predominantly hypomethylated signature; in contrast, mutations centered on the nuclear localization sequence resulted in a predominant hypermethylation signature. A subsequent study confirmed phenotypic differences between patients that correlated with the two episignatures [68].
Genes whose primary function is not associated with epigenetic and chromatin regulatory mechanisms, such as ubiquitin-conjugating enzyme E2 A (UBE2A) and spermine synthase (SMS) in X-linked syndromic forms of mental retardation-Nascimento and Snyder-Robinson types, respectively [44]-have also shown evidence of unique episignatures. The UBE2A gene at Xq24 encodes the RAD6 ubiquitin-conjugating enzyme and has been recently shown to be involved in histone modifications that control gene expression [69,70]. The SMS gene at Xp22.11 encodes for an enzyme involved in polyamine synthesis and recycling and is directly related to decarboxylated SAM. Previous studies have suggested that alterations in this polyamine synthesis could result in an excess of SAM and may lead to aberrant DNAm status [71].
Taken together, these studies show that DNAm episignatures can be detected in genes with various functions and provide strong evidence for the clinical utility of episignatures as diagnostic biomarkers in NDDs [43,44,72], while also enabling broader understanding of the clinical associations and biological roles of DNAm in genetic disorders.

Current Episignature Detection in NDDs
We recently described an approach to episignature mapping and development of a clinical EpiSign classifier in 65 genetic syndromes [43] involving bisulfite converted peripheral blood samples analyzed using methylation microarrays. Blood presents itself as the ideal tissue type for episignature development as it is a common clinical sample type and is easily accessible. Since episignatures represent a fundamental defect in NDDs caused by genetic variation in the germline, DNAm changes will be present in all subsequent tissues. This microarray technology enables a genome-wide, cost-effective, standardized, scalable and high throughput assessment of DNAm patterns, amenable to clinical validation in a diagnostic laboratory setting. This technology enables simultaneous assessment of up to 850,000 CpGs across the genome. By applying a custom bioinformatic pipeline to the methylation data obtained from these arrays, we are able to identify sensitive and disorder-specific episignatures. Using unsupervised machine learning models (MLMs), the sensitivity of an episignature can be assessed. Construction of multiclass supervised MLMs to compare a patient's DNAm data against controls and samples from other clinically validated episignatures at the same time, through the use of an expansive tissue-specific database, should be applied to confirm the specificity of a signature [44]. These methods rely on the ability to perform concurrent assessment of multiple disorders and controls, and highlights the importance of development of large-scale reference databases. To use episignatures in different tissues, a reference database would be required to establish the unique DNAm changes in the particular tissue that arise in development during differentiation and determine how this may impact the specific episignature. Our group has focused on episignatures in blood and has not assessed peripheral blood episignatures in other tissue types. Use of these supervised and unsupervised MLMs is also important when we consider the scalability of testing, as the list of episignatures continues to expand, requiring these algorithms to be capable of handling large amounts of data and computations in a cost-effective and timely fashion.
The ability to detect episignatures is highly contingent upon the intensity (effect size) and extent (number of differentially methylated CpGs) of the observed DNAm changes [44]. Some disorders, such as Sotos or TBRS, are associated with robust changes to the extent of involvement of tens of thousands of CpGs. In contrast, disorders such as the BAFopathies only exhibit a few hundred differentially methylated CpGs [64]. In light of this, sample size can play a role in the ability to detect episignatures in disorders associated with mild or moderate DNAm changes.
When analyzing methylation effects, it is important to consider confounding biological factors such as age, sex and blood cell composition, which are known to be associated with changes in methylation patterns in healthy individuals [73,74]. Methods should be in place to account for such factors when trying to decipher which observed methylation changes contribute to the underlying NDD.
To identify regions containing methylation changes, referred to as differentially methylated regions (DMRs), a 'bump hunting' approach [75] can be used, which typically considers regions containing 3-5 CpGs with greater than 10% methylation change between case samples and controls, and gaps of no more than 500 bp between neighboring CpGs [53]. DMRs can be useful in determining significant downstream effects of gene disruption and pathogenesis of disorders such as up-and down-regulated gene expression.
The complex bioinformatic pipeline required to identify episignatures, and to overcome previously mentioned confounding variables, relies heavily on a large-scale tissuespecific reference DNAm database, as well as bioinformatic and clinical genetic expertise [45]. Broadening utility of episignature assessment in the clinical setting involve screening of patients with suspected NDDs, as well as a functional assay for reclassification of VUSs.

The Use of Episignatures in the Diagnosis of NDDs
EpiSign is a clinical genome-wide DNAm test that has been available since 2019 that can detect over 60 disorders in more than 80 genes associated with Mendelian disorders through assessment of peripheral blood DNA [43]. A list of the current disorders detectable by EpiSign version 3 are listed in Table 1. In parallel with screening for episignatures, EpiSign also permits concurrent detection of FRX in males [37] and common imprinting disorders [76], thereby consolidating tests and reducing the need for additional reflexive testing [45]. In addition, its clinical utility in the assessment and reclassification of VUSs in genes with existing episignatures was recently reported in multiple studies [45,72,77,78]. EpiSign is the first and currently the only genome-wide DNAm clinical test offered for screening individuals with NDDs, and can be used as part of the diagnostic work up or for reclassification of VUSs. The reference EpiSign Knowledge Database (EKD) [44] utilized by the EpiSign assay contains thousands of peripheral blood DNAm profiles from both reference controls and NDD.
The cost of completing a methylation array is comparable to the cost of most CMAs, and the results are highly reproducible. The assay uses peripheral blood, similar to current CMA platforms, allowing streamlined adaption in the clinical setting given the overlap in equipment and laboratory techniques.
The ability to detect episignatures in patients with NDDs has been shown to increase the diagnostic yield, helping to resolve the diagnostic odyssey. A recent study assessing the clinical impact of EpiSign for patients with rare Mendelian disorders demonstrated a 27.6% diagnostic yield among patients with previous ambiguous/inconclusive genetic findings including genetic VUSs [45]. As episignature discovery expands and more disorders are added to the test repertoire, the diagnostic yield is likely to increase significantly, ultimately benefiting patients, their families and the related health care systems. This expansion in episignatures, however, may bring with it challenges, as it is likely to uncover syndromes with overlapping episignatures which may require the implementation of novel computational methods in order to classify these disorders.

Episignature Development in CNV-Associated Genomic Disorders Provides Insight into Pathological Mechanism
Changes in DNAm profiles, or episignatures, in patients with large CNV defects associated with genomic disorders have not been systematically studied, and it is plausible that large CNVs, much like gene specific variants, may exhibit unique diagnostic methylation signatures in patients with NDDs.
Our group recently published findings describing episignature discovery in patients with PHMDS [46], highlighting the novel insights DNA methylation analysis can contribute to the pathogenesis of CNV disorders. PHMDS is a genomic disorder associated with deletions of chromosome 22, involving partial or whole-gene disruption of the SH3 and multiple ankyrin repeat domains 3 gene (SHANK3). Intragenic variants in SHANK3 alone are responsible for a broad range of the phenotypic features observed in PHMDS [79]. However, this gene does not explain the entire phenotype in many patients, particularly speech and motor deficits, as well as renal abnormalities. The phenotypic variability and potential involvement of additional genes within the region has been previously assessed by multiple groups [80,81]. We demonstrated an episignature in patients with large deletions that was not observed in those with small deletions or SHANK3 gene level variants (Figure 1a-c). The minimal region of difference between these two deletion types, large versus small, included the bromodomain-containing protein 1 gene (BRD1), a gene involved in epigenetic mechanisms and a likely candidate gene for the methylation signature observed in these patients (Figure 1d). BRD1 is a component of a histone acetyltransferase complex that interacts with chromatin remodeling proteins and, before now, there was limited genotype-phenotype association reported in this gene. In addition, metabolic studies confirmed that these patients also exhibited very different metabolic profiles [46], further providing functional evidence for disease pathogenesis, as well as indicating targets for future therapies.

Defined Episignatures in Other CNV-Associated Genomic Disorders Provide Rationale to Further Expand Episignature Discovery
Symmetrical dose-dependent DNAm profiles have been reported in individuals with deletion of the 7q11.23 region (Williams syndrome; WS) or duplication of the same region (7q11.23 duplication syndrome) [83], highlighting the importance of DNAm in the pathogenesis of these disorders. This region contains a number of genes associated with epigenetic mechanisms, and a study by Aref-Eshghi et al. later showed that these methylation changes resulted in unique episignatures that could differentiate WS and 7q11.23 duplication syndrome from 40 other NDDs and congenital anomaly disorders [44]. In the same study, Aref-Eshghi et al. demonstrated another example of symmetrical DNAm pattern, this time when comparing Hunter-McAlpine syndrome (HMS) and Sotos syndrome. A distinct hypermethylation episignature is observed in HMS patients with duplications involving the 5q35 region containing the NSD1 gene, a direct contrast to the robust hypomethylation episignature seen in patients with Sotos syndrome, which is the result of loss of function variants in the same NSD1 gene [44].
A DNAm signature was reported in a cohort with the genomic disorder 16p11.2 deletion syndrome (16p11.2DS) [84]-a disorder associated with a variable phenotype that includes increased susceptibility to ASD. Several genes within this region play a role in histone or chromatin function; however, to date, no single candidate gene has been identified to be causative of this disorder or its resultant episignature. Moreover, 16p11.2DS shows reduced penetrance and variable expressivity, and although most deletions are de novo, many are inherited from apparently unaffected parents. These so-called "susceptibility CNVs" present challenges for clinicians in counselling families [41]. Due to the presence of a cluster of LCRs in this region that mediate CNVs through NAHR, there is a reciprocal duplication disorder (16p11.2 duplication syndrome) with similar diagnostic challenges. Studying methylation changes in patients with these susceptibility CNVs and their carrier parents could potentially unlock novel insights into the role of aberrant DNAm in reduced penetrance CNV disorders.
Our group recently described an aberrant DNAm pattern in patients with deletions of 12q24.31 encompassing the known histone modifier gene SET domain-containing protein 1B (SETD1B), and demonstrated that patients who harbored point mutations within SETD1B shared the same methylation episignature [78]. This study highlights that larger CNVs may exhibit the same methylation affects as gene specific variants within these regions.
The most common genomic disorder is a 22q11.2 deletion syndrome and is the result of a 1.5-3 Mb deletion also mediated by NAHR at a cluster of LCRs. Clinical manifestations of this disorder include DiGeorge and Velocardiofacial syndromes, and, to date, the phenotype-genotype relationship has not been fully elucidated. Through analysis of a cohort of individuals with 22q11.2 deletions, we identified an episignature that can differentiate 22q11.2 deletion syndrome from other NDDs on the clinical EpiSign test, including those considered in the differential diagnosis of this syndrome [85]. Among other findings, assessment of DMRs showed overlap with loci for orofacial clefting, a key phenotypic feature of this disorder. Through further analysis of atypical deletions and gene level variants, it may be possible to determine the gene, or genes, that play a role in the aberrant DNAm pattern observed, as well as insight into the mechanisms contributing to this disorder.
Only a few of the most prevalent genomic disorders have a candidate gene considered responsible for the entire phenotypic spectrum. Interestingly, where these candidate genes have been identified, they are predominantly involved in epigenetic regulation including chromatin remodeling or histone modification, e.g., CREBBP in Rubinstein-Taybi syndrome [86] and NSD1 in Sotos syndrome [87] (Table 2). Variants in most of these genes have already been assessed for genome-wide DNAm changes, and have been shown to exhibit unique and specific episignatures [43]. Overall, the majority of CNV disorders do not have a known or suspected candidate gene of interest. However, almost all of these regions contain one or more genes with epigenetic function (Table 2), e.g., chromodomain helicase DNA-binding protein 1-like (CHD1L) gene in 1q21.1 deletions and duplications, a gene that has a role in chromatin remodeling following DNA damage [88].  Taken together, the evidence suggests that CNV-associated genomic disorders may exhibit aberrant DNAm as the result of genes affected in their underlying deletions and duplications, especially when those regions include genes with epigenetic regulatory roles. CNV-associated genomic disorders are therefore strong candidates for episignature discovery. Investigating these syndromes further, including atypical CNVs and gene level variants within the same regions for possible sub-signatures, may uncover novel insights into the pathogenesis of these disorders. These studies may also identify new candidate genes responsible for some of the phenotypic presentation-should sub-signatures be uncovered for specific deleted or duplicated regions-and potentially unlock novel targets for more personalized treatment approaches.

Combined Detection of CNVs and DNA Methylation Episignatures in a Single Assay
Recent studies have shown it is possible to detect CNVs by applying computational methods to data obtained from DNAm arrays, such as the Illumina 450K and EPIC Bead Chip arrays [89][90][91]. Many of these pipelines are publicly available in Bioconductor, e.g., ChAMP [91,92], CopyNumber450k [93] and EpiCopy [89] (https://bioconductor.org/ packages/, accessed on 19 May 2022). The ability to integrate the detection of genetic and epigenetic findings can provide a more complete view of underlying pathogenic mechanisms.
We applied a similar computational approach using the DNAcopy package (Bioconductor.org) to our PHMDS cohort, and confirmed we could detect breakpoint coordinates similar to those obtained from conventional clinical CMA at the time of original diagnosis [46]; these findings are in line with previous studies [89][90][91][92][93].
Combining these detection methods is not without challenges, most notably in coverage of the genome, as CpG sites are not uniformly distributed throughout the genome and therefore methylation arrays lack the "backbone coverage" observed in high-density SNP arrays. However, it is plausible that, with modifications, a combined array could be developed containing a combination of copy number and CpG targeted probes to produce a clinically targeted array enabling accurate episignature and CNV analysis on a single platform. This has the potential to impact healthcare resource utilization by reducing concurrent testing in NDD patients, and decreasing the need for reflexive testing for disorders such as those associated with imprinting. There would continue to be limitations in the ability to detect low level mosaicism, as seen with existing CNV platforms; however, studies have shown the ability to detect mosaicism from methylation arrays in Kabuki syndrome 1 [94], imprinting disorders [76] and FRX [37].
Additional benefits of a combined testing platform include those to the patient; a combined array would permit screening for more disorders in a single assay, thereby potentially increasing diagnostic yield over that of the current first-tier clinical test (chromosome microarray), and shortening the time spent in the diagnostic odyssey. This approach could concurrently reduce the burden on clinical services and genetic counselling by providing results for CMA, FRX, imprinting and methylation in a single report, leading to a reduction in requisitions and clinic visits. A combined platform would also benefit oncology studies, where limitations in tumor sample availability can often impact research and diagnosis; this would permit the detection of CNVs and methylation status from the same volume of tissue as traditional testing.

Conclusions
The identification of episignatures in genomic disorders associated with CNVs could facilitate the expansion of screening capabilities for patients with NDDs, improving the diagnostic yield of clinical testing. This work may also provide novel insights into the pathogenesis of genomic disorders and provide targets for future therapies. The ability to combine CNV and episignature detection into a single assay would reduce the overall cost of testing, increase the number of disorders being screened for, and contribute to the reduction in alternative reflexive and concurrent genetic tests ordered. The benefit to patients and families in reducing wait times and increasing screened disorders, as well as reducing clinician and laboratory burden via fewer clinic visits and fewer genetic tests ordered, would have significant impacts on healthcare resource utilization and the costs associated with the diagnosis of NDDs.
Author Contributions: K.R. and B.S. conceived, performed the literature review and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-188).
Data Availability Statement: Not applicable.