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.

MSL2 variants lead to a neurodevelopmental syndrome with lack of coordination, epilepsy, specific dysmorphisms, and a distinct episignature.

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

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.

SRSF1 haploinsufficiency is responsible for a syndromic developmental disorder associated with intellectual disability.

SRSF1 (also known as ASF/SF2) is a non-small nuclear ribonucleoprotein (non-snRNP) that belongs to the arginine/serine (R/S) domain family. It recognizes and binds to mRNA, regulating both constitutive and alternative splicing. The complete loss of this proto-oncogene in mice is embryonically lethal. Through international data sharing, we identified 17 individuals (10 females and 7 males) with a neurodevelopmental disorder (NDD) with heterozygous germline SRSF1 variants, mostly de novo, including three frameshift variants, three nonsense variants, seven missense variants, and two microdeletions within region 17q22 encompassing SRSF1. Only in one family, the de novo origin could not be established. All individuals featured a recurrent phenotype including developmental delay and intellectual disability (DD/ID), hypotonia, neurobehavioral problems, with variable skeletal (66.7%) and cardiac (46%) anomalies. To investigate the functional consequences of SRSF1 variants, we performed in silico structural modeling, developed an in vivo splicing assay in Drosophila, and carried out episignature analysis in blood-derived DNA from affected individuals. We found that all loss-of-function and 5 out of 7 missense variants were pathogenic, leading to a loss of SRSF1 splicing activity in Drosophila, correlating with a detectable and specific DNA methylation episignature. In addition, our orthogonal in silico, in vivo, and epigenetics analyses enabled the separation of clearly pathogenic missense variants from those with uncertain significance. Overall, these results indicated that haploinsufficiency of SRSF1 is responsible for a syndromic NDD with ID due to a partial loss of SRSF1-mediated splicing activity.

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 detection of a strong episignature for Chung-Jansen syndrome, partially overlapping with Börjeson-Forssman-Lehmann and White-Kernohan syndromes.

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung-Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung-Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White-Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson-Forssman-Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung-Jansen, Börjeson-Forssman-Lehmann and White-Kernohan syndromes.

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 (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 data set 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.

Clinico-biological refinement of BCL11B-related disorder and identification of an episignature: A series of 20 unreported individuals.

PURPOSE: BCL11B-related disorder (BCL11B-RD) arises from rare genetic variants within the BCL11B gene, resulting in a distinctive clinical spectrum encompassing syndromic neurodevelopmental disorder, with or without intellectual disability, associated with facial features and impaired immune function. This study presents an in-depth clinico-biological analysis of 20 newly reported individuals with BCL11B-RD, coupled with a characterization of genome-wide DNA methylation patterns of this genetic condition. METHODS: Through an international collaboration, clinical and molecular data from 20 individuals were systematically gathered, and a comparative analysis was conducted between this series and existing literature. We further scrutinized peripheral blood DNA methylation profile of individuals with BCL11B-RD, contrasting them with healthy controls and other neurodevelopmental disorders marked by established episignature. RESULTS: Our findings unveil rarely documented clinical manifestations, notably including Rubinstein-Taybi-like facial features, craniosynostosis, and autoimmune disorders, all manifesting within the realm of BCL11B-RD. We refine the intricacies of T cell compartment alterations of BCL11B-RD, revealing decreased levels naive CD4+ T cells and recent thymic emigrants while concurrently observing an elevated proportion of effector-memory expressing CD45RA CD8+ T cells (TEMRA). Finally, a distinct DNA methylation episignature exclusive to BCL11B-RD is unveiled. CONCLUSION: This study serves to enrich our comprehension of the clinico-biological landscape of BCL11B-RD, potentially furnishing a more precise framework for diagnosis and follow-up of individuals carrying pathogenic BCL11B variant. Moreover, the identification of a unique DNA methylation episignature offers a valuable diagnosis tool for BCL11B-RD, thereby facilitating routine clinical practice by empowering physicians to reevaluate variants of uncertain significance within the BCL11B gene.

Microduplications of ARID1A and ARID1B cause a novel clinical and epigenetic distinct BAFopathy.

BACKGROUND: ARID1A/ARID1B haploinsufficiency leads to Coffin-Siris syndrome, duplications of ARID1A lead to a distinct clinical syndrome, whilst ARID1B duplications have not yet been linked to a phenotype. METHODS: We collected patients with duplications encompassing ARID1A and ARID1B duplications. RESULTS: 16 ARID1A and 13 ARID1B duplication cases were included with duplication sizes ranging from 0.1-1.2 Mb(1-44 genes) for ARID1A and 0.9-10.3 Mb(2-101 genes) for ARID1B. Both groups shared features, with ARID1A patients having more severe intellectual disability, growth delay and congenital anomalies. DNA methylation analysis showed that ARID1A patients had a specific methylation pattern in blood, which differed from controls and from patients with ARID1A or ARID1B loss-of-function variants. ARID1B patients appeared to have a distinct methylation pattern, similar to ARID1A duplication patients, but further research is needed to validate these results. Five cases with duplications including ARID1A or ARID1B initially annotated as duplications of uncertain significance were evaluated using PhenoScore and DNA methylation re-analysis, resulting in the reclassification of two ARID1A and two ARID1B duplications as pathogenic. CONCLUSION: Our findings reveal that ARID1B duplications manifest a clinical phenotype and ARID1A duplications have a distinct episignature that overlaps with that of ARID1B duplications, providing further evidence for a distinct and emerging BAFopathy caused by whole gene duplication rather than haploinsufficiency.

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.

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.

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature.

PURPOSE: Pathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD. METHODS: Through international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature. RESULTS: We recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism. CONCLUSION: Pathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.

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 correlation of genome-wide DNA methylation profiles in genetic neurodevelopmental disorders.

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive, and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes, which can share significant overlap among different conditions. In this study, we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders.

CDK13-related disorder: Report of a series of 18 previously unpublished individuals and description of an epigenetic signature.

PURPOSE: Rare genetic variants in CDK13 are responsible for CDK13-related disorder (CDK13-RD), with main clinical features being developmental delay or intellectual disability, facial features, behavioral problems, congenital heart defect, and seizures. In this paper, we report 18 novel individuals with CDK13-RD and provide characterization of genome-wide DNA methylation. METHODS: We obtained clinical phenotype and neuropsychological data for 18 and 10 individuals, respectively, and compared this series with the literature. We also compared peripheral blood DNA methylation profiles in individuals with CDK13-RD, controls, and other neurodevelopmental disorders episignatures. Finally, we developed a support vector machine-based classifier distinguishing CDK13-RD and non-CDK13-RD samples. RESULTS: We reported health and developmental parameters, clinical data, and neuropsychological profile of individuals with CDK13-RD. Genome-wide differential methylation analysis revealed a global hypomethylated profile in individuals with CDK13-RD in a highly sensitive and specific model that could aid in reclassifying variants of uncertain significance. CONCLUSION: We describe the novel features such as anxiety disorder, cryptorchidism, and disrupted sleep in CDK13-RD. We define a CDK13-RD DNA methylation episignature as a diagnostic tool and a defining functional feature of the evolving clinical presentation of this disorder. We also show overlap of the CDK13 DNA methylation profile in an individual with a functionally and clinically related CCNK-related disorder.

Episignature Mapping of TRIP12 Provides Functional Insight into Clark-Baraitser Syndrome.

Clark-Baraitser syndrome is a rare autosomal dominant intellectual disability syndrome caused by pathogenic variants in the TRIP12 (Thyroid Hormone Receptor Interactor 12) gene. TRIP12 encodes an E3 ligase in the ubiquitin pathway. The ubiquitin pathway includes activating E1, conjugating E2 and ligating E3 enzymes which regulate the breakdown and sorting of proteins. This enzymatic pathway is crucial for physiological processes. A significant proportion of TRIP12 variants are currently classified as variants of unknown significance (VUS). Episignatures have been shown to represent a powerful diagnostic tool to resolve inconclusive genetic findings for Mendelian disorders and to re-classify VUSs. Here, we show the results of DNA methylation episignature analysis in 32 individuals with pathogenic, likely pathogenic and VUS variants in TRIP12. We identified a specific and sensitive DNA methylation (DNAm) episignature associated with pathogenic TRIP12 variants, establishing its utility as a clinical biomarker for Clark-Baraitser syndrome. In addition, we performed analysis of differentially methylated regions as well as functional correlation of the TRIP12 genome-wide methylation profile with the profiles of 56 additional neurodevelopmental disorders.