ANKRD11 pathogenic variants and 16q24.3 microdeletions share an altered DNA methylation signature in patients with KBG syndrome.

Pathogenic variants in ANKRD11 or microdeletions at 16q24.3 are the cause of KBG syndrome (KBGS), a neurodevelopmental syndrome characterized by intellectual disability, dental and skeletal anomalies, and characteristic facies. The ANKRD11 gene encodes the ankyrin repeat-containing protein 11A transcriptional regulator, which is expressed in the brain and implicated in neural development. Syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show unique patterns of DNA methylation (DNAm) in peripheral blood, termed DNAm signatures. Given ANKRD11's role in chromatin modification, we tested whether pathogenic ANKRD11 variants underlying KBGS are associated with a DNAm signature. We profiled whole-blood DNAm in 21 individuals with ANKRD11 variants, 2 individuals with microdeletions at 16q24.3 and 28 typically developing individuals, using Illumina's Infinium EPIC array. We identified 95 differentially methylated CpG sites that distinguished individuals with KBGS and pathogenic variants in ANKRD11 (n = 14) from typically developing controls (n = 28). This DNAm signature was then validated in an independent cohort of seven individuals with KBGS and pathogenic ANKRD11 variants. We generated a machine learning model from the KBGS DNAm signature and classified the DNAm profiles of four individuals with variants of uncertain significance (VUS) in ANKRD11. We identified an intermediate classification score for an inherited missense variant transmitted from a clinically unaffected mother to her affected child. In conclusion, we show that the DNAm profiles of two individuals with 16q24.3 microdeletions were indistinguishable from the DNAm profiles of individuals with pathogenic variants in ANKRD11, and we demonstrate the diagnostic utility of the new KBGS signature by classifying the DNAm profiles of individuals with VUS in ANKRD11.

Suleiman-El-Hattab syndrome: a histone modification disorder caused by TASP1 deficiency.

BACKGROUND: TASP1 encodes an endopeptidase activating histone methyltransferases of the KMT2 family. Homozygous loss-of-function variants in TASP1 have recently been associated with Suleiman-El-Hattab syndrome. We report six individuals with Suleiman-El-Hattab syndrome and provide functional characterization of this novel histone modification disorder in a multi-omics approach. METHODS: Chromosomal microarray/exome sequencing in all individuals. Western blotting from fibroblasts in two individuals. RNA sequencing and proteomics from fibroblasts in one individual. Methylome analysis from blood in two individuals. Knock-out of tasp1 orthologue in zebrafish and phenotyping. RESULTS: All individuals had biallelic TASP1 loss-of-function variants and a phenotype including developmental delay, multiple congenital anomalies (including cardiovascular and posterior fossa malformations), a distinct facial appearance and happy demeanor. Western blot revealed absence of TASP1. RNA sequencing/proteomics showed HOX gene downregulation (HOXA4, HOXA7, HOXA1 and HOXB2) and dysregulation of transcription factor TFIIA. A distinct methylation profile intermediate between control and Kabuki syndrome (KMT2D) profiles could be produced. Zebrafish tasp1 knock-out revealed smaller head size and abnormal cranial cartilage formation in tasp1 crispants. CONCLUSION: This work further delineates Suleiman-El-Hattab syndrome, a recognizable neurodevelopmental syndrome. Possible downstream mechanisms of TASP1 deficiency include perturbed HOX gene expression and dysregulated TFIIA complex. Methylation pattern suggests that Suleiman-El-Hattab syndrome can be categorized into the group of histone modification disorders including Wiedemann-Steiner and Kabuki syndrome.

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature.

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

DNA methylation signatures for chromatinopathies: current challenges and future applications.

Pathogenic variants in genes that encode epigenetic regulators are the cause for more than 100 rare neurodevelopmental syndromes also termed "chromatinopathies". DNA methylation signatures, syndrome-specific patterns of DNA methylation alterations, serve as both a research avenue for elucidating disease pathophysiology and a clinical diagnostic tool. The latter is well established, especially for the classification of variants of uncertain significance (VUS). In this perspective, we describe the seminal DNA methylation signature research in chromatinopathies; the complex relationships between genotype, phenotype and DNA methylation, and the future applications of DNA methylation signatures.

The utility of DNA methylation signatures in directing genome sequencing workflow: Kabuki syndrome and CDK13-related disorder.

Kabuki syndrome (KS) is a neurodevelopmental disorder characterized by hypotonia, intellectual disability, skeletal anomalies, and postnatal growth restriction. The characteristic facial appearance is not pathognomonic for KS as several other conditions demonstrate overlapping features. For 20-30% of children with a clinical diagnosis of KS, no causal variant is identified by conventional genetic testing of the two associated genes, KMT2D and KDM6A. Here, we describe two cases of suspected KS that met clinical diagnostic criteria and had a high gestalt match on the artificial intelligence platform Face2Gene. Although initial KS testing was negative, genome-wide DNA methylation (DNAm) was instrumental in guiding genome sequencing workflow to establish definitive molecular diagnoses. In one case, a positive DNAm signature for KMT2D led to the identification of a cryptic variant in KDM6A by genome sequencing; for the other case, a DNAm signature different from KS led to the detection of another diagnosis in the KS differential, CDK13-related disorder. This approach illustrates the clinical utility of DNAm signatures in the diagnostic workflow for the genome analyst or clinical geneticist-especially for disorders with overlapping clinical phenotypes.

Potential pathophysiological role of microRNA 193b-5p in human placentae from pregnancies complicated by preeclampsia and intrauterine growth restriction.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy complications resulting from abnormal placental development. MicroRNAs can regulate placental development and contribute to disease, by influencing gene expression. Our previous study revealed an increase in miR-193b-5p expression in placentae from patients with early-onset pregnancy complications and identified candidate gene targets for miR-193b-5p. The purpose of this study is two-fold, first to validate candidate gene targets predicted for miR-193b-5p from microRNA-RNA expression data. Second, to overexpress miR-193b-5p in a trophoblast cell line (HTR-8/SVneo) to assess impact on trophoblast cell proliferation and migration. Integration of the miRNA and RNA sequencing expression data revealed 10 candidate gene targets for miR-193b-5p across all patient groups (PE only, IUGR only, PE + IUGR). Luciferase experiments identified two gene targets for miR-193b-5p, APLN and FGF13. Real-time PCR confirmed a median 45% decrease of FGF13 expression across 3 patient groups, and 50% decrease of APLN expression in patients with PE + IUGR. Following transfection of HTR-8/SVneo cells with miR-193b-5p mimics, APLN and FGF13 mRNA expression in HTR-8/SVneo was reduced by a median percentage of 30% and 45%, respectively. Concomitantly, HTR-8/SVneo cells demonstrate 40% reduction in cell migration. APLN and FGF13 immunoreactivity was identified strongly in the cytotrophoblast cells of the human placentae. These findings suggest that miR-193b-5p may contribute to trophoblast dysfunction observed in pregnancy complications such as PE and IUGR.

Creation of matrix protein gene variants of two serotypes of vesicular stomatitis virus as prime-boost vaccine vectors.

UNLABELLED: To take advantage of live recombinant vesicular stomatitis viruses (rVSVs) as vaccine vectors for their high yield and for their induction of strong and long-lasting immune responses, it is necessary to make live vaccine vectors safe for use without losing their immunogenicity. We have generated safer and highly efficient recombinant VSV vaccine vectors by combining the M51R mutation in the M gene of serotype VSV-Indiana (VSVInd) with a temperature-sensitive mutation (tsO23) of the VSVInd Orsay strain. In addition, we have generated two new serotype VSV-New Jersey (VSVNJ) vaccine vectors by combining M48R and M51R mutations with G22E and L110F mutations in the M gene, rVSVNJ(G22E M48R M51R) [rVSVNJ(GMM)] and VSVNJ(G22E M48R M51R L110F) [rVSVNJ(GMML)]. The combined mutations G21E, M51R, and L111F in the M protein of VSVInd significantly reduced the burst size of the virus by up to 10,000-fold at 37°C without affecting the level of protein expression. BHK21 cells and SH-SY5Y human neuroblastoma cells infected with rVSVInd(GML), rVSVNJ(GMM), and rVSVNJ(GMML) showed significantly reduced cytopathic effects in vitro at 37°C, and mice injected with 1 million infectious virus particles of these mutants into the brain showed no neurological dysfunctions or any other adverse effects. In order to increase the stability of the temperature-sensitive mutant, we have replaced the phenylalanine with alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting L111A mutant showed the temperature-sensitive phenotype of rVSVInd(GML) and increased stability. Twenty consecutive passages of rVSVInd(GML) with an L111A mutation did not convert back to leucine (UUG) at position 111 in the M protein gene. IMPORTANCE: Recombinant vesicular stomatitis viruses as live vaccine vectors are very effective in expressing foreign genes and inducing adaptive T cell and B cell immune responses. As with any other live viruses in humans or animals, the use of live rVSVs as vaccine vectors demands the utmost safety. Our strategy to attenuate rVSVInd by utilizing a temperature-sensitive assembly-defective mutation of L111A and combining it with an M51R mutation in the M protein of rVSVInd significantly reduced the pathogenicity of the virus while maintaining highly effective virus production. We believe our new temperature-sensitive M gene mutant of rVSVInd(GML) and M gene mutants of rVSVNJ(GMM) and rVSVNJ(GMML) add excellent vaccine vectors to the pool of live viral vectors.

Biological relevance of CNV calling methods using familial relatedness including monozygotic twins.

BACKGROUND: Studies involving the analysis of structural variation including Copy Number Variation (CNV) have recently exploded in the literature. Furthermore, CNVs have been associated with a number of complex diseases and neurodevelopmental disorders. Common methods for CNV detection use SNP, CNV, or CGH arrays, where the signal intensities of consecutive probes are used to define the number of copies associated with a given genomic region. These practices pose a number of challenges that interfere with the ability of available methods to accurately call CNVs. It has, therefore, become necessary to develop experimental protocols to test the reliability of CNV calling methods from microarray data so that researchers can properly discriminate biologically relevant data from noise. RESULTS: We have developed a workflow for the integration of data from multiple CNV calling algorithms using the same array results. It uses four CNV calling programs: PennCNV (PC), Affymetrix® Genotyping Console™ (AGC), Partek® Genomics Suite™ (PGS) and Golden Helix SVS™ (GH) to analyze CEL files from the Affymetrix® Human SNP 6.0 Array™. To assess the relative suitability of each program, we used individuals of known genetic relationships. We found significant differences in CNV calls obtained by different CNV calling programs. CONCLUSIONS: Although the programs showed variable patterns of CNVs in the same individuals, their distribution in individuals of different degrees of genetic relatedness has allowed us to offer two suggestions. The first involves the use of multiple algorithms for the detection of the largest possible number of CNVs, and the second suggests the use of PennCNV over all other methods when the use of only one software program is desirable.

Copy number variation distribution in six monozygotic twin pairs discordant for schizophrenia.

We have evaluated copy number variants (CNVs) in six monozygotic twin pairs discordant for schizophrenia. The data from Affymetrix® Human SNP 6.0 arrays™ were analyzed using Affymetrix® Genotyping Console™, Partek® Genomics Suite™, PennCNV, and Golden Helix SVS™. This yielded both program-specific and overlapping results. Only CNVs called by Affymetrix Genotyping Console, Partek Genomics Suite, and PennCNV were used in further analysis. This analysis included an assessment of calls in each of the six twin pairs towards identification of unique CNVs in affected and unaffected co-twins. Real time polymerase chain reaction (PCR) experiments confirmed one CNV loss at 7q11.21 that was found in the affected patient but not in the unaffected twin. The results identified CNVs and genes that were previously implicated in mental abnormalities in four of the six twin pairs. It included PYY (twin pairs 1 and 5), EPHA3 (twin pair 3), KIAA1211L (twin pair 4), and GPR139 (twin pair 5). They represent likely candidate genes and CNVs for the discordance of four of the six monozygotic twin pairs for this heterogeneous neurodevelopmental disorder. An explanation for these differences is ontogenetic de novo events that differentiate in the monozygotic twins during development.

A new blood DNA methylation signature for Koolen-de Vries syndrome: Classification of missense KANSL1 variants and comparison to fibroblast cells.

Pathogenic variants in KANSL1 and 17q21.31 microdeletions are causative of Koolen-de Vries syndrome (KdVS), a neurodevelopmental syndrome with characteristic facial dysmorphia. Our previous work has shown that syndromic conditions caused by pathogenic variants in epigenetic regulatory genes have identifiable patterns of DNA methylation (DNAm) change: DNAm signatures or episignatures. Given the role of KANSL1 in histone acetylation, we tested whether variants underlying KdVS are associated with a DNAm signature. We profiled whole-blood DNAm for 13 individuals with KANSL1 variants, four individuals with 17q21.31 microdeletions, and 21 typically developing individuals, using Illumina's Infinium EPIC array. In this study, we identified a robust DNAm signature of 456 significant CpG sites in 8 individuals with KdVS, a pattern independently validated in an additional 7 individuals with KdVS. We also demonstrate the diagnostic utility of the signature and classify two KANSL1 VUS as well as four variants in individuals with atypical clinical presentation. Lastly, we investigated tissue-specific DNAm changes in fibroblast cells from individuals with KdVS. Collectively, our findings contribute to the understanding of the epigenetic landscape related to KdVS and aid in the diagnosis and classification of variants in this structurally complex genomic region.

Multiomics of Bohring-Opitz syndrome truncating ASXL1 mutations identify canonical and noncanonical Wnt signaling dysregulation.

ASXL1 (additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo protein-truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS; OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, distinctive facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies, including heart defects and severe skeletal defects giving rise to a typical BOS posture. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia. We used primary cells from individuals with BOS (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multiomics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data show that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, which encodes a planar cell polarity pathway protein that acts through noncanonical Wnt signaling to direct tissue patterning and cell migration. This multiomics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.

Generation of DNA Methylation Signatures and Classification of Variants in Rare Neurodevelopmental Disorders Using EpigenCentral.

There are more than 700 genes that encode proteins that function in epigenetic regulation and chromatin modification. Germline variants in these genes (typically heterozygous) are associated with rare neurodevelopmental disorders (NDDs) characterized by growth abnormalities and intellectual and developmental delay. Advancements in next-generation sequencing have dramatically increased the detection of pathogenic sequence variants in genes encoding epigenetic machinery associated with NDDs and, concurrently, the number of clinically uninterpretable variants classified as variants of uncertain significance (VUS). Recently, DNA methylation (DNAm) signatures, disorder-specific patterns of DNAm change, have emerged as a functional tool that provides insights into disorder pathophysiology and can classify pathogenicity of variants in NDDs. To date, our group and others have identified DNAm signatures for more than 60 Mendelian neurodevelopmental disorders caused by variants in genes encoding epigenetic machinery. There is broad interest in both the research and clinical communities to develop and catalog DNAm signatures in rare NDDs, but there are challenges in optimizing study design considerations and availability of platforms that integrate bioinformatics tools with the appropriate statistical framework required to analyze genome-wide DNAm data. We previously published EpigenCentral, a platform for analysis of DNAm data in rare NDDs. In this article, we utilize the published Weaver syndrome dataset to provide step-by-step protocols for using EpigenCentral for exploratory analysis to identify DNAm signatures and for classification of NDD variants. We also provide important considerations for experimental design and interpretation of DNAm results. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Exploratory analysis to identify disorder-specific DNAm signatures Basic Protocol 2: Classification of variants associated with neurodevelopmental disorders.

DNA methylation signature associated with Bohring-Opitz syndrome: a new tool for functional classification of variants in ASXL genes.

The additional sex combs-like (ASXL) gene family-encoded by ASXL1, ASXL2, and ASXL3-is crucial for mammalian development. Pathogenic variants in the ASXL gene family are associated with three phenotypically distinct neurodevelopmental syndromes. Our previous work has shown that syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show consistent patterns of genome-wide DNA methylation (DNAm) alterations, i.e., DNAm signatures in peripheral blood. Given the role of ASXL1 in chromatin modification, we hypothesized that pathogenic ASXL1 variants underlying Bohring-Opitz syndrome (BOS) have a unique DNAm signature. We profiled whole-blood DNAm for 17 ASXL1 variants, and 35 sex- and age-matched typically developing individuals, using Illumina's Infinium EPIC array. We identified 763 differentially methylated CpG sites in individuals with BOS. Differentially methylated sites overlapped 323 unique genes, including HOXA5 and HOXB4, supporting the functional relevance of DNAm signatures. We used a machine-learning classification model based on the BOS DNAm signature to classify variants of uncertain significance in ASXL1, as well as pathogenic ASXL2 and ASXL3 variants. The DNAm profile of one individual with the ASXL2 variant was BOS-like, whereas the DNAm profiles of three individuals with ASXL3 variants were control-like. We also used Horvath's epigenetic clock, which showed acceleration in DNAm age in individuals with pathogenic ASXL1 variants, and the individual with the pathogenic ASXL2 variant, but not in individuals with ASXL3 variants. These studies enhance our understanding of the epigenetic dysregulation underpinning ASXL gene family-associated syndromes.

Exposure to Gestational Diabetes Mellitus (GDM) alters DNA methylation in placenta and fetal cord blood.

BACKGROUND: Fetal exposure to maternal GDM increases offspring risk for adult-onset metabolic syndromes. Epigenetic modifications such as DNA methylation are modulators for fetal metabolic programming and susceptibility to adult-onset disease. This study investigates genome-wide DNA methylation in GDM exposed cord blood and placenta. METHODS: Oral glucose tolerance testing between 24 and 28 weeks of pregnancy was used to determine severity of glucose intolerance. We measured DNA methylation (DNAm) using the Illumina Infinium 450 K array in 42 fetal cord blood and 36 placenta samples. RESULTS: We identified 662 and 99 CpG sites in GDM placenta and cord blood, respectively at p-value <0.01 and a methylation difference >5% after adjustment for confounders. Annotated sites for AHRR and PTPRN2 were common to cord blood and placenta. Adding published GDM cord blood DNAm data we segregated patients based on treatment (Diet Only vs. +Insulin) and identified altered CpG sites to be unique to each GDM treatment group. CONCLUSION: Consistency in findings with other studies provides evidence for the role of DNAm in placental and fetal responses to hyperglycemia. However, segregating DNAm analysis of GDM samples based on treatment may help delineate whether observed DNAm alterations are reflective of adaptive responses or treatment effects in utero.

Identification of miR-210-5p in human placentae from pregnancies complicated by preeclampsia and intrauterine growth restriction, and its potential role in the pregnancy complications.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy complications resulting from abnormal placental development. As epigenetic regulators, microRNAs can regulate placental development and contribute to the disease pathophysiology by influencing the expression of genes involved in placental development or disease. Our previous study revealed an increase in miR-210-5p expression in placentae from patients with early-onset pregnancy complications and identified candidate gene targets for miR-210-5p. The purpose of this study was to: (i) validate candidate gene targets predicted for miR-210-5p from microRNA-RNA expression data, and (ii) overexpress miR-210-5p in a trophoblast cell line (HTR-8/SVneo) to assess impact on trophoblast cell functions. Integration of the miRNA and RNA sequencing expression data revealed 8 candidate gene targets for miR-210-5p in patients with PE only or PE + IUGR. Luciferase reporter assays identified two gene targets for miR-210-5p, CSF1 and ITGAM. Real-time PCR confirmed the decreased expression of CSF1 and ITGAM in patients with PE + IUGR. Immunohistochemistry of placentae from late second trimester identified CSF1 and ITGAM in intermediate trophoblast cells in the decidua. Expression levels of CSF1 and ITGAM were reduced in HTR-8/SVneo cells following increased miR-210-5p expression. Concomitantly, HTR-8/SVneo cells demonstrate an average 45% reduction in cell migration. These findings suggest that miR-210-5p may contribute to dysfunction of intermediate trophoblasts and potentially contribute to the disease process of these pregnancy complications.

Placental microRNAs in pregnancies with early onset intrauterine growth restriction and preeclampsia: potential impact on gene expression and pathophysiology.

BACKGROUND: A normally developed placenta is integral to a successful pregnancy. Preeclampsia (PE) and intrauterine growth restriction (IUGR) are two common pregnancy related complications that maybe a result of abnormal placental development. Placental microRNAs (miRNAs) have been investigated as potential biomarkers for these complications, as they may play a role in placental development and pathophysiology by influencing gene expression. The purpose of this study is to utilize next-generation sequencing to determine miRNA and gene expression in human placental (chorionic villous) samples from three distinct patient groups with early-onset (EO) PE, IUGR, or PE + IUGR. METHODS: Placental tissues were collected from four patient groups (control [N = 21], EO-PE [N = 20], EO-IUGR [N = 18], and EO-PE + IUGR [N = 20]), and total RNA was used for miRNA and RNA sequencing on the Illumina Hiseq2000 platform. For stringent differential expression analysis multiple analysis programs were used to analyze both expression datasets in each patient group compared to gestational age-matched controls. RESULTS: Analysis revealed miRNAs and genes that are disease-specific, as well as others that were common between disease groups, which suggests common underlying placental pathologies in EO-PE and EO-IUGR. More specifically, 6 miRNAs and 22 genes were identified to be differentially expressed in all three patient groups. In addition, integrative analysis between the miRNA and gene expression datasets revealed candidate gene targets for miRNAs of interest. CONCLUSIONS: Integration of miRNA and RNA profiling in the same three subgroups of pregnancy complications, provides an alternate level of molecular information, in addition it can be used to better understand both unique and common molecular mechanisms involved in the pathophysiology of these diseases.