Haploinsufficiency of ZFHX3, encoding a key player in neuronal development, causes syndromic intellectual disability.

Neurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function (LoF) variation in ZFHX3 as a cause for syndromic intellectual disability (ID). ZFHX3 is a zinc-finger homeodomain transcription factor involved in various biological processes, including cell differentiation and tumorigenesis. We describe 42 individuals with protein-truncating variants (PTVs) or (partial) deletions of ZFHX3, exhibiting variable intellectual disability and autism spectrum disorder, recurrent facial features, relative short stature, brachydactyly, and, rarely, cleft palate. ZFHX3 LoF associates with a specific methylation profile in whole blood extracted DNA. Nuclear abundance of ZFHX3 increases during human brain development and neuronal differentiation. ZFHX3 was found to interact with the chromatin remodeling BRG1/Brm-associated factor complex and the cleavage and polyadenylation complex, suggesting a function in chromatin remodeling and mRNA processing. Furthermore, ChIP-seq for ZFHX3 revealed that it predominantly binds promoters of genes involved in nervous system development. We conclude that loss-of-function variants in ZFHX3 are a cause of syndromic ID associating with a specific DNA methylation profile.

Identification of a DNA methylation episignature for recurrent constellations of embryonic malformations.

The term "recurrent constellations of embryonic malformations" (RCEM) is used to describe a number of multiple malformation associations that affect three or more body structures. The causes of these disorders are currently unknown, and no diagnostic marker has been identified. Consequently, providing a definitive diagnosis in suspected individuals is challenging. In this study, genome-wide DNA methylation analysis was conducted on DNA samples obtained from the peripheral blood of 53 individuals with RCEM characterized by clinical features recognized as VACTERL and/or oculoauriculovertebral spectrum association. We identified a common DNA methylation episignature in 40 out of the 53 individuals. Subsequently, a sensitive and specific binary classifier was developed based on the DNA methylation episignature. This classifier can facilitate the use of RCEM episignature as a diagnostic biomarker in a clinical setting. The study also investigated the functional correlation of RCEM DNA methylation relative to other genetic disorders with known episignatures, highlighting the common genomic regulatory pathways involved in the pathophysiology of RCEM.

Identification of a robust DNA methylation signature for Fanconi anemia.

Fanconi anemia (FA) is a clinically variable and genetically heterogeneous cancer-predisposing disorder representing the most common bone marrow failure syndrome. It is caused by inactivating predominantly biallelic mutations involving >20 genes encoding proteins with roles in the FA/BRCA DNA repair pathway. Molecular diagnosis of FA is challenging due to the wide spectrum of the contributing gene mutations and structural rearrangements. The assessment of chromosomal fragility after exposure to DNA cross-linking agents is generally required to definitively confirm diagnosis. We assessed peripheral blood genome-wide DNA methylation (DNAm) profiles in 25 subjects with molecularly confirmed clinical diagnosis of FA (FANCA complementation group) using Illumina's Infinium EPIC array. We identified 82 differentially methylated CpG sites that allow to distinguish subjects with FA from healthy individuals and subjects with other genetic disorders, defining an FA-specific DNAm signature. The episignature was validated using a second cohort of subjects with FA involving different complementation groups, documenting broader genetic sensitivity and demonstrating its specificity using the EpiSign Knowledge Database. The episignature properly classified DNA samples obtained from bone marrow aspirates, demonstrating robustness. Using the selected probes, we trained a machine-learning model able to classify EPIC DNAm profiles in molecularly unsolved cases. Finally, we show that the generated episignature includes CpG sites that do not undergo functional selective pressure, allowing diagnosis of FA in individuals with reverted phenotype due to gene conversion. These findings provide a tool to accelerate diagnostic testing in FA and broaden the clinical utility of DNAm profiling in the diagnostic setting.

Blepharophimosis with intellectual disability and Helsmoortel-Van Der Aa Syndrome share episignature and phenotype.

Blepharophimosis with intellectual disability (BIS) is a recently recognized disorder distinct from Nicolaides-Baraister syndrome that presents with distinct facial features of blepharophimosis, developmental delay, and intellectual disability. BIS is caused by pathogenic variants in SMARCA2, that encodes the catalytic subunit of the superfamily II helicase group of the BRG1 and BRM-associated factors (BAF) forming the BAF complex, a chromatin remodeling complex involved in transcriptional regulation. Individuals bearing variants within the bipartite nuclear localization (BNL) signal domain of ADNP present with the neurodevelopmental disorder known as Helsmoortel-Van Der Aa Syndrome (HVDAS). Distinct DNA methylation profiles referred to as episignatures have been reported in HVDAS and BAF complex disorders. Due to molecular interactions between ADNP and BAF complex, and an overlapping craniofacial phenotype with narrowing of the palpebral fissures in a subset of patients with HVDAS and BIS, we hypothesized the possibility of a common phenotype-specific episignature. A distinct episignature was shared by 15 individuals with BIS-causing SMARCA2 pathogenic variants and 12 individuals with class II HVDAS caused by truncating pathogenic ADNP variants. This represents first evidence of a sensitive phenotype-specific episignature biomarker shared across distinct genetic conditions that also exhibit unique gene-specific episignatures.

The missing link: ARID1B non-truncating variants causing Coffin-Siris syndrome due to protein aggregation.

ARID1B is the most frequently mutated gene in Coffin-Siris syndrome (CSS). To date, the vast majority of causative variants reported in ARID1B are truncating, leading to nonsense-mediated mRNA decay. In the absence of experimental data, only few ARID1B amino acid substitutions have been classified as pathogenic, mainly based on clinical data and their de novo occurrence, while most others are currently interpreted as variants of unknown significance. The present study substantiates the pathogenesis of ARID1B non-truncating/NMD-escaping variants located in the SMARCA4-interacting EHD2 and DNA-binding ARID domains. Overexpression assays in cell lines revealed that the majority of EHD2 variants lead to protein misfolding and formation of cytoplasmic aggresomes surrounded by vimentin cage-like structures and co-localizing with the microtubule organisation center. ARID domain variants exhibited not only aggresomes, but also nuclear aggregates, demonstrating robust pathological effects. Protein levels were not compromised, as shown by quantitative western blot analysis. In silico structural analysis predicted the exposure of amylogenic segments in both domains due to the nearby variants, likely causing this aggregation. Genome-wide transcriptome and methylation analysis in affected individuals revealed expression and methylome patterns consistent with those of the pathogenic haploinsufficiency ARID1B alterations in CSS cases. These results further support pathogenicity and indicate two approaches for disambiguation of such variants in everyday practice. The few affected individuals harbouring EHD2 non-truncating variants described to date exhibit mild CSS clinical traits. In summary, this study paves the way for the re-evaluation of previously unclear ARID1B non-truncating variants and opens a new era in CSS genetic diagnosis.

Identification of DNA methylation episignature for the intellectual developmental disorder, autosomal dominant 21 syndrome, caused by variants in the CTCF gene.

PURPOSE: The main objective of this study was to assess clinical features and genome-wide DNA methylation profiles in individuals affected by intellectual developmental disorder, autosomal dominant 21 (IDD21) syndrome, caused by variants in the CCCTC-binding factor (CTCF) gene. METHODS: DNA samples were extracted from peripheral blood of 16 individuals with clinical features and genetic findings consistent with IDD21. DNA methylation analysis was performed using the Illumina Infinium Methylation EPIC Bead Chip microarrays. The methylation levels were fitted in a multivariate linear regression model to identify the differentially methylated probes. A binary support vector machine classification model was constructed to differentiate IDD21 samples from controls. RESULTS: We identified a highly specific, reproducible, and sensitive episignature associated with CTCF variants. Six variants of uncertain significance were tested, of which 2 mapped to the IDD21 episignature and clustered alongside IDD21 cases in both heatmap and multidimensional scaling plots. Comparison of the genomic DNA methylation profile of IDD21 with that of 56 other neurodevelopmental disorders provided insights into the underlying molecular pathophysiology of this disorder. CONCLUSION: The robust and specific CTCF/IDD21 episignature expands the growing list of neurodevelopmental disorders with distinct DNA methylation profiles, which can be applied as supporting evidence in variant classification.

Discovery of DNA methylation signature in the peripheral blood of individuals with history of antenatal exposure to valproic acid.

PURPOSE: Valproic acid or valproate is an effective antiepileptic drug; however, embryonic exposure to valproate can result in a teratogenic disorder referred to as fetal valproate syndrome (FVS, OMIM #609442). Currently there are no diagnostic biomarkers for the condition. This study aims to define an episignature biomarker for teratogenic antenatal exposure to valproate. METHODS: DNA extracted from peripheral blood of individuals with teratogenic antenatal exposure to valproate was processed using DNA methylation microarrays. Subsequently, methylation profiling and construction of support vector machine classifiers were performed in R. RESULTS: Genomic DNA methylation analysis was applied, and a distinct DNA methylation profile was identified in the majority of affected individuals. This profile was used to develop a diagnostic episignature classifier. The valproate exposure episignature exhibited high sensitivity and specificity relative to a large reference dataset of unaffected controls and individuals with a wide spectrum of syndromic disorders with episignatures. Gene set enrichment analysis demonstrated an enrichment for terms associated with cell adhesion, including significant overrepresentation of the cadherin superfamily. CONCLUSION: This study provides evidence of a robust peripheral blood-based diagnostic epigenetic biomarker for a prenatal teratogenic disorder.

DNA methylation episignature, extension of the clinical features, and comparative epigenomic profiling of Hao-Fountain syndrome caused by variants in USP7.

PURPOSE: Hao-Fountain syndrome (HAFOUS) is a neurodevelopmental disorder caused by pathogenic variants in USP7. HAFOUS is characterized by developmental delay, intellectual disability, speech delay, behavioral abnormalities, autism spectrum disorder, seizures, hypogonadism, and mild dysmorphic features. We investigated the phenotype of 18 participants with HAFOUS and performed DNA methylation (DNAm) analysis, aiming to generate a diagnostic biomarker. Furthermore, we performed comparative analysis with known episignatures to gain more insight into the molecular pathophysiology of HAFOUS. METHODS: We assessed genomic DNAm profiles of 18 individuals with pathogenic variants and variants of uncertain significance (VUS) in USP7 to map and validate a specific episignature. The comparison between the USP7 cohort and 56 rare genetic disorders with earlier reported DNAm episignatures was performed with statistical and functional correlation. RESULTS: We mapped a sensitive and specific DNAm episignature for pathogenic variants in USP7 and utilized this to reclassify the VUS. Comparative epigenomic analysis showed evidence of HAFOUS similarity to a number of other rare genetic episignature disorders. CONCLUSION: We discovered a sensitive and specific DNAm episignature as a robust diagnostic biomarker for HAFOUS that enables VUS reclassification in USP7. We also expand the phenotypic spectrum of 9 new and 5 previously reported individuals with HAFOUS.

Diagnostic utility and reporting recommendations for clinical DNA methylation episignature testing in genetically undiagnosed rare diseases.

PURPOSE: This study aims to assess the diagnostic utility and provide reporting recommendations for clinical DNA methylation episignature testing based on the cohort of patients tested through the EpiSign Clinical Testing Network. METHODS: The EpiSign assay utilized unsupervised clustering techniques and a support vector machine-based classification algorithm to compare each patient's genome-wide DNA methylation profile with the EpiSign Knowledge Database, yielding the result that was reported. An international working group, representing distinct EpiSign Clinical Testing Network health jurisdictions, collaborated to establish recommendations for interpretation and reporting of episignature testing. RESULTS: Among 2399 cases analyzed, 1667 cases underwent a comprehensive screen of validated episignatures, imprinting, and promoter regions, resulting in 18.7% (312/1667) positive reports. The remaining 732 referrals underwent targeted episignature analysis for assessment of sequence or copy-number variants (CNVs) of uncertain significance or for assessment of clinical diagnoses without confirmed molecular findings, and 32.4% (237/732) were positive. Cases with detailed clinical information were highlighted to describe various utility scenarios for episignature testing. CONCLUSION: Clinical DNA methylation testing including episignatures, imprinting, and promoter analysis provided by an integrated network of clinical laboratories enables test standardization and demonstrates significant diagnostic yield and clinical utility beyond DNA sequence analysis in rare diseases.

Integrated omics analyses clarifies ATRX copy number variant of uncertain significance.

Partial duplications of genes can be challenging to detect and interpret and, therefore, likely represent an underreported cause of human disease. X-linked dominant variants in ATRX are associated with Alpha-thalassemia/impaired intellectual development syndrome, X-linked (ATR-X syndrome), a clinically heterogeneous disease generally presenting with intellectual disability, hypotonia, characteristic facies, genital anomalies, and alpha-thalassemia. We describe an affected male with a de novo hemizygous intragenic duplication of ~43.6 kb in ATRX, detected by research genome sequencing following non-diagnostic clinical testing. RNA sequencing and DNA methylation episignature analyses were central in variant interpretation, and this duplication was subsequently interpreted as disease-causing. This represents the smallest reported tandem duplication within ATRX associated with disease. This case demonstrates the diagnostic utility of integrating multiple omics technologies, which can ultimately lead to a definitive diagnosis for rare disease patients.

Refining the 9q34.3 microduplication syndrome reveals mild neurodevelopmental features associated with a distinct global DNA methylation profile.

Precise regulation of gene expression is important for correct neurodevelopment. 9q34.3 deletions affecting the EHMT1 gene result in a syndromic neurodevelopmental disorder named Kleefstra syndrome. In contrast, duplications of the 9q34.3 locus encompassing EHMT1 have been suggested to cause developmental disorders, but only limited information has been available. We have identified 15 individuals from 10 unrelated families, with 9q34.3 duplications <1.5 Mb in size, encompassing EHMT1 entirely. Clinical features included mild developmental delay, mild intellectual disability or learning problems, autism spectrum disorder, and behavior problems. The individuals did not consistently display dysmorphic features, congenital anomalies, or growth abnormalities. DNA methylation analysis revealed a weak DNAm profile for the cases with 9q34.3 duplication encompassing EHMT1, which could segregate the majority of the affected cases from controls. This study shows that individuals with 9q34.3 duplications including EHMT1 gene present with mild non-syndromic neurodevelopmental disorders and DNA methylation changes different from Kleefstra syndrome.

Development of a comprehensive approach to adult hereditary cancer testing in Ontario.

BACKGROUND: Genetic testing for hereditary cancer susceptibility has advanced over time due to the discovery of new risk genes, improved technology and decreased cost. In the province of Ontario, testing eligibility criteria were initially developed to include hereditary breast, ovarian and colorectal cancer syndromes. The rapid evolution of genetic technologies has facilitated the ability to interrogate a large number of genes concurrently. This, coupled with new knowledge about risk genes, necessitated a coordinated approach to expanding the scope of genes and indications tested and synchronisation of access and test utilisation across the province as required in a publicly funded universal healthcare system. METHODS: Ontario Health-Cancer Care Ontario convened expert working groups to develop a standardised and comprehensive cancer gene list for adults and accompanying hereditary cancer testing (HCT) criteria using an evidence-based framework and broad laboratory and clinical genetics engagement. RESULTS: A standardised 76-cancer-gene panel, organised into 13 larger disease site panels and 25 single/small gene panels, was developed and endorsed by the working groups. Provincial genetic testing eligibility criteria were updated to align with the new panels and to guide clinical decision-making. In the first year following the implementation of these changes, 10 564 HCT panels were performed with an overall mutation detection rate of 12.2%. CONCLUSION: Using an evidence framework and broad clinical engagement to develop and endorse an updated guidance document, cancer genetic testing for adults in Ontario is now standardised and coordinated across the province.

Integration of EpiSign, facial phenotyping, and likelihood ratio interpretation of clinical abnormalities in the re-classification of an ARID1B missense variant.

Heterozygous ARID1B variants result in Coffin-Siris syndrome. Features may include hypoplastic nails, slow growth, characteristic facial features, hypotonia, hypertrichosis, and sparse scalp hair. Most reported cases are due to ARID1B loss of function variants. We report a boy with developmental delay, feeding difficulties, aspiration, recurrent respiratory infections, slow growth, and hypotonia without a clinical diagnosis, where a previously unreported ARID1B missense variant was classified as a variant of uncertain significance. The pathogenicity of this variant was refined through combined methodologies including genome-wide methylation signature analysis (EpiSign), Machine Learning (ML) facial phenotyping, and LIRICAL. Trio exome sequencing and EpiSign were performed. ML facial phenotyping compared facial images using FaceMatch and GestaltMatcher to syndrome-specific libraries to prioritize the trio exome bioinformatic pipeline gene list output. Phenotype-driven variant prioritization was performed with LIRICAL. A de novo heterozygous missense variant, ARID1B p.(Tyr1268His), was reported as a variant of uncertain significance. The ACMG classification was refined to likely pathogenic by a supportive methylation signature, ML facial phenotyping, and prioritization through LIRICAL. The ARID1B genotype-phenotype has been expanded through an extended analysis of missense variation through genome-wide methylation signatures, ML facial phenotyping, and likelihood-ratio gene prioritization.

DNA methylation episignature and comparative epigenomic profiling of HNRNPU-related neurodevelopmental disorder.

PURPOSE: HNRNPU haploinsufficiency is associated with developmental and epileptic encephalopathy 54. This neurodevelopmental disorder is characterized by developmental delay, intellectual disability, speech impairment, and early-onset epilepsy. We performed genome-wide DNA methylation (DNAm) analysis in a cohort of individuals to develop a diagnostic biomarker and gain functional insights into the molecular pathophysiology of HNRNPU-related disorder. METHODS: DNAm profiles of individuals carrying pathogenic HNRNPU variants, identified through an international multicenter collaboration, were assessed using Infinium Methylation EPIC arrays. Statistical and functional correlation analyses were performed comparing the HNRNPU cohort with 56 previously reported DNAm episignatures. RESULTS: A robust and reproducible DNAm episignature and global DNAm profile were identified. Correlation analysis identified partial overlap and similarity of the global HNRNPU DNAm profile to several other rare disorders. CONCLUSION: This study demonstrates new evidence of a specific and sensitive DNAm episignature associated with pathogenic heterozygous HNRNPU variants, establishing its utility as a clinical biomarker for the expansion of the EpiSign diagnostic test.

DNA methylation episignature for Witteveen-Kolk syndrome due to SIN3A haploinsufficiency.

PURPOSE: Witteveen-Kolk syndrome (WITKOS) is a rare, autosomal dominant neurodevelopmental disorder caused by heterozygous loss-of-function alterations in the SIN3A gene. WITKOS has variable expressivity that commonly overlaps with other neurodevelopmental disorders. In this study, we characterized a distinct DNA methylation epigenetic signature (episignature) distinguishing WITKOS from unaffected individuals as well as individuals with other neurodevelopmental disorders with episignatures and described 9 previously unpublished individuals with SIN3A haploinsufficiency. METHODS: We studied the phenotypic characteristics and the genome-wide DNA methylation in the peripheral blood samples of 20 individuals with heterozygous alterations in SIN3A. A total of 14 samples were used for the identification of the episignature and building of a predictive diagnostic biomarker, whereas the diagnostic model was used to investigate the methylation pattern of the remaining 6 samples. RESULTS: A predominantly hypomethylated DNA methylation profile specific to WITKOS was identified, and the classifier model was able to diagnose a previously unresolved test case. The episignature was sensitive enough to detect individuals with varying degrees of phenotypic severity carrying SIN3A haploinsufficient variants. CONCLUSION: We identified a novel, robust episignature in WITKOS due to SIN3A haploinsufficiency. This episignature has the potential to aid identification and diagnosis of individuals with WITKOS.

A Rational Approach to JAK2 Mutation Testing in Patients with Elevated Hemoglobin: Results from the JAK2 Prediction Cohort (JAKPOT) Study.

BACKGROUND: Erythrocytosis, most often measured as an increase in hemoglobin and/or hematocrit, is a common reason for referral to internal medicine and hematology clinics and a rational approach is required to effectively identify patients with polycythemia vera while avoiding over-investigation. AIM: We aimed to develop and validate a simple rule to predict JAK2 mutation positivity based on complete blood count parameters to aid in the diagnostic approach to patients referred for elevated hemoglobin. SETTING: Internal medicine and hematology clinics at an academic tertiary referral center. PARTICIPANTS: The JAK2 Prediction Cohort (JAKPOT), a large retrospective cohort (n = 901) of patients evaluated by internal medicine and hematology specialists for elevated hemoglobin. DESIGN: JAK2 mutation analysis was performed in all patients and clinical and laboratory variables were collected. Patients were randomly divided into derivation and validation cohorts. A prediction rule was developed using data from the derivation cohort and tested in the validation cohort. KEY RESULTS: The JAKPOT prediction rule included three variables: (i) red blood cell count >6.45×1012/L, (ii) platelets >350×109/L, and (iii) neutrophils >6.2×109/L; absence of all criteria was effective at ruling out JAK2-positivity with sensitivities 94.7% and 100%, and negative predictive values of 98.8% and 100% in the derivation and validation cohorts, respectively, with an overall low false negative rate of 0.4%. The rule was validated for three different methods of JAK2 testing. Applying this rule to our entire cohort would have resulted in over 50% fewer tests. CONCLUSION: In patients with elevated hemoglobin, the use of a simple prediction rule helps to accurately identify patients with a low likelihood of having a JAK2 mutation, potentially limiting costly over-investigation in this common referral population.

Leber Hereditary Optic Neuropathy in Southwestern Ontario: A Growing List of Mutations.

BACKGROUND: Leber hereditary optic neuropathy (LHON) is a rare but bilaterally blinding disease. Three characteristic disease-causing point mutations, and other less common mutations, are most often found on the mitochondrially encoded genes of NADH-ubiquinone oxidoreductase core subunits (MT-ND). The purpose of this study is to provide an overview of LHON mutations in Southwestern Ontario and to describe the associated demographic and clinical characteristics. METHODS: A retrospective genetic and clinical chart review was performed from January 2015 to 2020. Patients were identified within a mitochondrial mutation database and included if a mutation was detected on the MT-ND1, -ND4, or -ND6 genes. A clinical chart review was done on all available patients. RESULTS: Forty-five of 63 patients identified had classic disease-causing mutations (6.7% m.3460G>A, 44.4% m.11778G>A, and 48.9% m.14484T>C). Several of the remaining 18 patients had rare mutations previously documented in association with LHON. Of the 14 patients with clinical charts accessible for review, 12 had symptomatic disease, and all but one had bilateral optic neuropathies. Nine patients had classic LHON mutations and 3 had possible novel mutations; 7 were males; 9 had final visual acuity ≤ 20/200 in at least one eye; and 6 of those had ≤20/400 in both eyes. CONCLUSIONS: This study adds to the literature on LHON in Canada, and specifically Southwestern Ontario. The demographic and clinical data regarding LHON in this geographic location, as well as possible novel disease-causing mutations, provide important information to aid clinicians in recognizing cases of LHON that may otherwise be disregarded.

Complexity in Genetic Epilepsies: A Comprehensive Review.

Epilepsy is a highly prevalent neurological disorder, affecting between 5-8 per 1000 individuals and is associated with a lifetime risk of up to 3%. In addition to high incidence, epilepsy is a highly heterogeneous disorder, with variation including, but not limited to the following: severity, age of onset, type of seizure, developmental delay, drug responsiveness, and other comorbidities. Variable phenotypes are reflected in a range of etiologies including genetic, infectious, metabolic, immune, acquired/structural (resulting from, for example, a severe head injury or stroke), or idiopathic. This review will focus specifically on epilepsies with a genetic cause, genetic testing, and biomarkers in epilepsy.

Functional Insight into and Refinement of the Genomic Boundaries of the JARID2-Neurodevelopmental Disorder Episignature.

JARID2 (Jumonji, AT-rich interactive domain 2) haploinsufficiency is associated with a clinically distinct neurodevelopmental syndrome. It is characterized by intellectual disability, developmental delay, autistic features, behavior abnormalities, cognitive impairment, hypotonia, and dysmorphic features. JARID2 acts as a transcriptional repressor protein that is involved in the regulation of histone methyltransferase complexes. JARID2 plays a role in the epigenetic machinery, and the associated syndrome has an identified DNA methylation episignature derived from sequence variants and intragenic deletions involving JARID2. For this study, our aim was to determine whether patients with larger deletions spanning beyond JARID2 present a similar DNA methylation episignature and to define the critical region involved in aberrant DNA methylation in 6p22-p24 microdeletions. We examined the DNA methylation profiles of peripheral blood from 56 control subjects, 13 patients with (likely) pathogenic JARID2 variants or patients carrying copy number variants, and three patients with JARID2 VUS variants. The analysis showed a distinct and strong differentiation between patients with (likely) pathogenic variants, both sequence and copy number, and controls. Using the identified episignature, we developed a binary model to classify patients with the JARID2-neurodevelopmental syndrome. DNA methylation analysis indicated that JARID2 is the driver gene for aberrant DNA methylation observed in 6p22-p24 microdeletions. In addition, we performed analysis of functional correlation of the JARID2 genome-wide methylation profile with the DNA methylation profiles of 56 additional neurodevelopmental disorders. To conclude, we refined the critical region for the presence of the JARID2 episignature in 6p22-p24 microdeletions and provide insight into the functional changes in the epigenome observed when regulation by JARID2 is lost.

Functional annotation of genomic variation: DNA methylation episignatures in neurodevelopmental Mendelian disorders.

The breadth and complexity of genetic testing in patients with suspected Mendelian neurodevelopmental disorders has rapidly expanded in the past two decades. However, in spite of advances in genomic technologies, genetic diagnosis remains elusive in more than half of these patients. Epigenomics, and in particular genomic DNA methylation profiles, are now known to be associated with the underpinning genetic defects in a growing number of Mendelian disorders. These often highly specific and sensitive molecular biomarkers have been used to screen these patient populations, resolve ambiguous clinical cases and interpret genetic variants of unknown clinical significance. Increasing the diagnostic yield beyond genomic sequencing technologies has rapidly propelled epigenomics to clinical utilization, with recent introduction of DNA methylation 'EpiSign' analysis in clinical diagnostic laboratories. This review provides an overview of the principles, applications and limitations of DNA methylation episignature analysis in patients with neurodevelopmental Mendelian disorders, and discusses clinical implications of this emerging diagnostic technology.