Ehmt2 Loss-of-function Alterations Cause a Kleefstra-like Syndrome

Ehmt2 Loss-of-function Alterations Cause a Kleefstra-like Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ehmt2 Loss-of-function Alterations Cause a Kleefstra-like Syndrome Maria Barrero, Beatriz Martínez-Delgado, Estrella López-Martín, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3893528/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The dysregulation of the epigenetic machinery has been linked to neurodevelopmental defects in humans. One such syndrome is Kleefstra syndrome (KS), which results from heterozygous alterations in the EHMT1 gene, leading to loss of function. EHMT1 and EHMT2 are highly similar histone methyltransferases that play crucial roles in development. Despite their similarity, alterations in EHMT2 have not been previously reported. In this study, we present a pediatric patient exhibiting a phenotype overlapping with KS, harboring a de novo single base substitution in EHMT2. This substitution results in the amino acid change p.Ala1077Ser in the catalytic SET domain, causing a decrease in the affinity of this domain for histone H3 tail and a three- to five-fold reduction in enzyme activity. As part of an advanced diagnostic strategy, we leveraged epigenomics and proteomics data to comprehensively characterize the EHMT2 p.Ala1077Ser variant. Analysis of DNA methylation, histone methylation, and gene expression profiles reveals a substantial overlap between the EHMT2 p.Ala1077Ser variant and KS. Based on these findings, we propose that EHMT2 haploinsufficiency leads to a Kleefstra-like syndrome. While we cannot entirely rule out dominant negative effects caused by the EHMT2 p.Ala1077Ser variant, our data, in conjunction with previously published studies, suggest that the loss of EHMT2 function is more detrimental to cells than the loss of EHMT1. This may explain the rarity of individuals with alterations in EHMT2 . rare diseases EHMT2 Kleefstra syndrome clinical epigenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Dysregulation of the epigenetic machinery is associated with neurodevelopmental defects in humans. Kleefstra syndrome (KS) is a neurodevelopmental syndrome caused by heterozygous deletions at chromosome 9q34.3 that include the EHMT1 gene (~ 50%) or heterozygous intragenic EHMT1 pathogenic variants (~ 50%) 1,2 . In addition, loss-of-function mutations in the KMT2C (MLL3) gene cause a Kleefstra-like syndrome called Kleefstra syndrome-2 (KLEFS2) 3 – 5 . Moreover, although very rare, de novo mutations in epigenetic regulators genes MBD5 , MLL3 , SMARCB1 , and NR1I3 have been associated with intellectual disability disorders that fall into the Kleefstra syndrome’s phenotypic spectrum 3 . EHMT1 (also known as GLP) and EHMT2 (also known as G9a) are highly homologous proteins that contain a SET domain that confers them the ability to mono- and dimethylate lysine 9 at histone H3 (H3K9) 6 , 7 . H3K9 methylation is finely regulated by several methyltransferases and demethylases and plays important roles in development, differentiation, and cancer 8 . EHMT1 and EHMT2 can form homo- or heterodimers through SET domain interactions, being heterodimers the most active configuration and the preferred form in cells 9 . Both Ehmt1-/- and Ehmt2-/- mice show severe developmental delays and early embryonic lethality that correlates with lower levels of H3k9me1 and me2 9,10 . Despite the overlap in domains and functions of EHMT1 and EHMT2 and their relevance for development, patients with alterations in EHMT2 have never been reported. Here we report a Kleefstra-like syndrome case with a missense variant in the catalytic SET domain of EHMT2 that causes alanine 1077 change to serine reducing its affinity for the H3 tail and the activity of the enzyme by three- to five-fold. Results from our study allow defining this variant as pathogenic following the ACMG criteria 11 , since it appears the novo in a patient with disease and no family history (PS2), in vitro functional studies are supportive of a damaging effect (PS3), it is absent from controls in GnomAD (PM2) and the variant is located in a critical functional domain (PM1). MATERIALS AND METHODS Participants Patients ND095 and ND120 were recruited by the undiagnosed rare diseases program SpainUDP 12 at the Institute of Rare Diseases Research (IIER), Spanish National Institute of Health Carlos III (ISCIII). Peripheral blood samples and skin biopsies were collected from patients and their parents to perform trio-based whole-exome sequencing and establishment of fibroblast cultures respectively. Whole Exome sequencing Whole exome sequencing and data analysis was performed on the probands and their unaffected parents. Genomic DNA was extracted from peripheral blood using the Qiagen QIAamp DNA kit. Whole Exome Sequencing (WES) libraries were prepared using the Nimblegen MedExome + ChrMit as enrichment kit and HS2000 v4, 2x100bp sequencing platform in the ND095 family and Nextera Felx DNA Library Prep and Illumina NextSeq500 in the ND120 family. Data analysis was performed using two different standardized protocols as previously described 12 . These included an in-house analysis and a parallel analysis using the Genomic Analysis module of the RD-Connect Genome-Phenome Analysis Platform (GPAP) 13 . All commonly identified rare variants were further analysed, checking all available scientific evidence through detailed searches in public databases (including Gene-Card, NCBI, UniProt, OMIM, PubMed and ExAc). Sanger sequencing was performed to validate candidate variants using the following primers for EHMT2 5´-AGCAGGGTAAGGAGGGTCTC-3’ and 5´-CCACCTCCTAATAGCCCACA-3’, and for EHMT1 5’-CTTCTTCTCTGTGGGGCGAG-3´ and 5´-CACCATAAGCATCAGCATCAGC-3´ Fibroblast culture Human dermal fibroblasts cultures were established from skin tissue samples. Briefly, fibroblasts were mechanically isolated by dissecting the dermal layer of the skin and the resulting fragments were incubated at 37°C in Dulbeccos' modified Eagle's medium (DMEM) containing 2% of fetal calf serum. Cells were expanded by incubation in 75 cm2 culture flasks at 37°C with 5% CO2 and 95% humidity in DMEM containing 10% fetal bovine serum. RNA-seq analysis Total RNA was extracted from fibroblasts using the RNeasy mini kit from Qiagen. RNA-seq was performed at BGI Tech Solutions with two or three biological replicates per condition. Briefly, ribosomal RNAs were removed using a RNase H-based method and first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP, end repair, A addition and adaptor ligation. The U-labeled second-strand template was digested with Uracil-DNA-Glycosylase (UDG) and amplified by PCR. The resulting library was validated by quality control. The PCR products were then heat denatured and circularized by the splint oligo sequence to generate a single strand circle DNA followed by rolling circle replication to create DNA nanoballs (DNB) for sequencing on the MGI DNBSEQ platform. Raw sequencing data with adapter sequences or low-quality sequences was trimmed or filtered and examined by FastQC for basic quality controls. The sequencing analysis was carried out using Galaxy ( https://usegalaxy.eu/ ). Paired reads were aligned to the human hg19 genome build using STAR 14 . Gene counts were calculated using HTseq-count 15 and differential expression between patients and healthy fibroblast was interrogated using DEseq 16 . Differentially expressed genes were considered at an adjusted p-value < 0.05. For enrichment analysis in differentially expressed genes, we used GSEA preranked 17 with genes ranked according to p-value corrected log2 of fold change. Interrogated gene sets were Gene Ontology Biological Process (GOBP) and transcription factor targets (TFT). In addition, we performed enrichment in GOBP terms in significantly upregulated or downregulated genes using Panther ( https://pantherdb.org/ ) 18 . Scatterplots showing correlations were generated with ggplot2 in Galaxy. Pearson’s correlation and bubble plots were calculated using SRPlot 19 . Network representation of enriched GOBP terms was generated using Cytoscape Enrichment Map and AutoAnotate Apps 20 . Production of recombinant proteins pET28a-LIC containing the catalytic domain of human EHMT2 (aa913-1193) was a gift from Cheryl Arrowsmith (Addgene plasmid # 25503; http://n2t.net/addgene:25503 ; RRID:Addgene_25503). Mutation p.Ala1077Ser was introduced using the QuikChange II XL Site-Directed Mutagenesis Kit from Agilent. Expression and purification of His-tagged EHMT2 catalytic domain WT and p.Ala1077Ser in bacteria has been previously described 21 . Recombinant H3 was expressed in E. coli and purified as previously described 22 Histone methyltransferase assays Histone methyltransferase assays were performed using the fluorescence-based assay SAM Methyltransferase Assay SAMfluoro™ from G-Biosciences that detects the formation of resorufin over time as a result of the methyltransferase reaction. Assays were performed using 250, 500 or 1000 ng of His-tagged WT or p.Ala1077Ser EHMT2 catalytic domains and 2.5 µg of recombinant histone H3 following the manufacturer instructions. Cumulative reads were measured every minute for 45 minutes. Specific activity was calculated using a resorufin standard. Histone peptide binding assays 10 µg of biotinylated histone 3 peptides aa 1 to 21 (Active Motif) unmodified, monomethylated at K9 or dimethylated at K9 were incubated overnight at 4ºC with 5 ug of recombinant EHMT2 wild type or p.Ala1077Ser mutant catalytic domain in 300 µl of binding buffer 50 mM Tris pH 7.5, 150 mM NaCl, 0.05% NP-40 and 1 mM PMSF. After 1 hour incubation with Pierce streptavidin magnetic beads and extensive washing with binding buffer bound proteins were resolved by SDS-PAGE and stained with Coomassie blue. Gels were scanned using the Bio-Rad GelDoc Go Imaging System and bands quantified using ImageJ 23 . Protein modeling PDB Protein Data Bank entry 5JJ0 ( https://doi.org/10.2210/pdb5JJ0/pdb ) corresponding to the catalytic domain of EHMT2 complexed with the histone peptide H3K9M and SAM was explored in 3D using PDB resources 24 . EpiSign assay Methylation analysis was conducted using the clinically validated EpiSign assay, following previously established methods 25 – 28 . Methylated and unmethylated signal intensities generated from the EPIC array were imported into R 3.5.1 for normalization, background correction, and filtering. Beta values were then calculated as a measure of methylation level, ranging from 0 (no methylation) to 1 (complete methylation), and processed through the established support vector machine (SVM) classification algorithm for EpiSign disorders. The classifier utilized the EpiSign Knowledge Database, which consists of over 10,000 methylation profiles from reference disorder-specific and unaffected control cohorts, to generate disorder-specific methylation variant pathogenicity (MVP) scores. These MVP scores are a measure of prediction confidence for each disorder and range from 0 (discordant) to 1 (highly concordant). A positive classification typically generates MVP scores greater than 0.5. The final matched EpiSign result is generated using these scores, along with the assessment of hierarchical clustering and multidimensional scaling. Detection of histone modifications by mass spectrometry Mass spectrometry was performed by Active Motif. Histones were acid extracted from a cell pellet containing 2.5x10 6 cells, derivatized via propionylation, digested with trypsin, newly formed N-termini were propionylated as previously described 29 . Histones were extracted by incubating samples at room temperature for 1 hour in 0.2M sulfuric acid with intermittent vortexing. Histones were then precipitated by the addition of trichloroacetic acid (TCA) on ice, and recovered by centrifugation at 10,000 x g for 5 minutes at 4°C. The pellet was then washed once with 1mL cold acetone/0.1% HCl and twice with 100% acetone, and then air dried in a clean hood. The histones were propionylated by adding 1:3 v/v propionic anhydride/2-propanol and incrementally adding ammonium hydroxide to keep the pH around 8, and subsequently dried in a SpeedVac concentrator. The pellet was then resuspended in 100 mM ammonium bicarbonate and adjusted to pH 7–8 with ammonium hydroxide. The histones were then digested with trypsin, resuspended in 100mM ammonium bicarbonate overnight at 37°C, and dried in a SpeedVac concentrator. The pellet was resuspended in 100mM ammonium bicarbonate and propionylated a second time by adding 1:3 v/v propionic anhydride/2-propanol and incrementally adding ammonium hydroxide to keep the pH around 8, and subsequently dried in a SpeedVac concentrator. Histone peptides were resuspended in 50 µL of 0.1% TFA and 3 µl were injected with 3 technical replicates in a Thermo Scientific TSQ Quantum Ultra mass spectrometer coupled with an UltiMate 3000 Dionex nano-liquid chromatography system. The data was quantified using Skyline 30 , and represents the percent of each modification within the total pool of that amino acid residue. RESULTS Clinical characteristics of the proband overlap with Kleefstra syndrome Proband ND095 was remitted to the undiagnosed rare diseases program SpainUDP 12 at the age of 4.8 years. Despite exhaustive clinical and genetic studies, mainly through candidate gene panels, the patient had remained undiagnosed until that time. The proband is a male that suffered from intrauterine growth restriction and congenital anomalies, including coarctation of the aorta and renal medullar cystic disease. At the age of 2 years, he was diagnosed with global developmental delay, characterized by slow developing of gross motor milestones and expressive language delay, hypotonia without weakness and nephrocalcinosis. At the craniofacial level, he had brachycephaly, plagiocephaly with very flat occiput, broad face and midface hypoplasia, synophrys, sparse medial eyebrows, epicanthus, lateral deviation of upslanted palpebral fissures, mildly everted lower eyelids, palpebral ptosis (right eye), anteverted nares, smooth philtrum and carp-like mouth. In addition, he had dental diastema, microdontia, persistent fetal fingertip pads and scoliosis (Fig. 1 A and Supplementary File 1). From the clinical point of view, the patient’s phenotype overlaps significantly with the clinical characteristics of KS (Table 1 ). Table 1 Anomalies found in patient ND095 compared to Kleefstra syndrome 1 , 2 , 44 , 46 . Anomalies ND095 Kleefstra High birth weight - 8–50% Microcephaly - 30–80% Brachycephaly + 33% Flat face + 16% Midface hypoplasia + 55–100% Hypertelorism + 55–70% Arched eyebrows + 27% Synophrys + 55–80% Upslanting palpebral fissures + 30–70% Epicanthal folds + 30–70% Ocular anomalies - 45% Anteverted nares + 40–80% Carp mouth + 77% Protruding tongue - 40–60% Dental anomalies + 10–15% Ear anomalies - 45–80% Hypoacusia - 20–30% Cardiovascular anomalies + 40–45% Umbilical/inguinal hernia + 15–20% Renal issues + 15–30% Genital anomalies + 45–50% of males Skeletal anomalies + 30–50% Limb anomalies + 70% Brachydactyly + 16% Short stature - 10–39% Obesity - 30–40% Hypotonia + 60–80% Psychomotor delay/intellectual disability + 100% Speech anomalies non-verbal ~ 100% Autism ASD - 30–75% Behavioral problems stereotypy 65–70% Sleep disorder - 20–50% Brain imaging anomalies partial empty sella turcica 50–60% Epilepsy one episode 20–50% Gastro-esophageal reflux - 14–19% Tracheomalacia - 5–11% Respiratory complications + 5–14% Recurrent infections - 26–64% Whole-exome sequencing revealed a variant of uncertain significance (VUS) in the EHMT2 gene Whole-exome sequencing of patient ND095 and his parents, carried out at SpainUDP, revealed no detectable deleterious variants in the KS causing gene EHMT1 . In addition, no alterations were detected in KMT2C , which causes Kleefstra syndrome 2 5 . Similarly, no pathogenic variants were detected in chromatin-related genes MBD5 , MLL3 , SMARCB1 , and NR1I3 , which have been previously associated with Kleefstra-like syndromes 3 . Surprisingly, the sequencing revealed a heterozygous de novo variant in the EHMT2 gene (hg19 chr6:31848838C > A; NM_006709.5:c.3229G > T) that resulted in the amino acid change alanine 1077 to serine (p.Ala1077Ser) (Fig. 1 B). This variant was not present in GnomAD V4 genomes nor exomes, and neither was it reported in ClinVar. Several in silico predictors classified this variant as pathogenic (EIGEN, FATHMM, LRT, MutPred MVP, REVEL) while it was classified as a VUS by others (Mutation Taster, Mutation Assessor, SIFT, PROVEAN). The p.Ala1077Ser variant decreases the activity of EHMT2 Ala1077 is located in the catalytic SET domain of EHMT2, in a highly conserved region among human histone methyltransferases (Fig. 1 C). Ala1077 is, however, far from the AdoMet cofactor interacting amino acids (Fig. 1 C) and the presumed dimerization region (Fig. 1 D). The published EHMT2 catalytic domain structure in complex with H3 tail 31 shows that Ala1077 interacts with the H3 tail residue threonine 6, suggesting that it might affect the catalytic activity of EHMT2 through changes in this interaction (Fig. 1 E). To test this possibility, we first evaluated the effects of the p.Ala1077Ser variant in the activity of the enzyme. We used an in vitro fluorescence-based assay that allows the detection of methyltransferase activity over time to test the activity of both wild type and Ala1077Ser EHMT2 catalytic domains expressed and purified in E. coli , on recombinant histone H3. Figures 2 A and 2 B shows that the p.Ala1077Ser variant drastically reduces the methyltransferase activity of EHMT2 on histone H3 over time and the specific activity of the enzyme by five-fold, respectively. Replication of the histone methyltransferase assay using different EHMT2 concentrations shows that the Ala1077Ser variant reduces the specific activity of the enzyme by three- to five-fold (Fig. 2 B, Supplementary Fig. 2). Next, we evaluated if the p.Ala1077Ser variant affects the interaction of EHMT2 with the histone H3 tail by testing the interaction of both EHMT2 wild type and the p.Ala1077Ser mutant with synthetic H3 peptides (aa 1–21), either unmethylated, mono or dimethylated at lysine 9, in an in vitro pull-down assay. Figures 2 C and 2 D show that the p.Ala1077Ser variant significantly reduces de affinity of the catalytic domain for the histone H3 tail. In addition, we detected a reduced affinity of the wild type catalytic domain for the dimethylated H3 peptide compared to unmethylated or monomethylated peptides. These results are in line with previous data that suggest a loss of affinity of methyltransferases for their substrate once it is fully methylated 21 , 32 . This behavior was not observed in the mutant catalytic domain (Figs. 2 C and D). The Episign test classifies patient ND095 as Kleefstra syndrome. In order to investigate the overlap of patient ND095 with KS specific DNA methylation signature, we compared the blood DNA methylation profile of patient ND095 with Kleefstra patients using version three of the test EpiSign 26 . This test, based in blood DNA methylation profiles, allows the diagnosis of over fifty different genetic diseases. Unsupervised hierarchical clustering of DNA methylation profiles shows that ND095 clusters with Kleefstra syndrome patients and clearly separates from healthy controls (Fig. 3 A). The computational model classified patient ND095 as KS with high confidence, generating a low score for other 56 syndromes (Fig. 3 C). However, the principal component analysis of DNA methylation showed that ND095 had a particular methylation profile that differs slightly from KS patients (Fig. 3 B). This data suggests the involvement of an EHMT1 -related but different causal gene in the phenotype of patient ND095. Overlap of molecular signatures between patient ND095 and Kleefstra syndrome. Next, we established fibroblasts cultures from a healthy donor, the ND095 patient carrying the EHMT2 p.Ala1077Ser variant and a patient (ND120) recently diagnosed with KS through our program, who carries a frameshift variant in EHMT1 (NM_024757.5:c.1881delT p.His620ThrfsTer12), and quantified histone modifications by mass spectrometry. Figure 4 A shows that ND095 fibroblasts had decreased levels of mono-, di- and trimethylated H3K9, while levels of H3K27me3 remained unchanged. In correlation, we found an increase in unmodified and acetylated H3K9. Similar but less dramatic effects were found in KS patient ND120 (Fig. 4 A). These results are in agreement with a previous characterization of changes in histone modifications after knockdown of both EHMT2 and EHMT1 33 and are compatible with the loss of EHMT2 activity in patient ND095. Afterwards, we carried out RNA-seq in the fibroblasts cultures and identified differentially expressed genes (DEGs) in ND095 and ND120 fibroblasts compared to healthy control fibroblasts (Supplementary Table 1). Among DEGs we found both upregulated and downregulated genes, with a higher number of DEGs in ND095 compared to ND120 (Fig. 4 B). This correlates with the more severe effects on H3K9 methylation in ND095 fibroblasts found by mass spectrometry. Despite this difference, there was a significant overlap in down and upregulated genes between ND095 and ND120 (Fig. 4 B) and a significant correlation comparing the fold change of expression of all genes (Fig. 4 C). We next investigated the enrichment of GOBP terms in the DEGs using gene set enrichment analysis (GSEA). Results show a significant enrichment of genes involved in the morphogenesis of diverse tissues and organs derived from the three embryonic layers, and genes involved in the extracellular matrix in genes upregulated in ND095 compared to control fibroblasts (Fig. 4 D). Significant enrichment in morphogenesis genes in upregulated genes was also identified in patient ND120 (Supplementary Fig. 3A) and in genes commonly upregulated in both patients (Supplementary Fig. 3B). Genes downregulated in ND095 fibroblasts compared to healthy control fibroblasts were mainly involved in cell cycle (Fig. 4 D). Additionally, a significant enrichment of E2F transcription factors binding sites was found around the transcriptional start site of genes downregulated in ND095 fibroblasts (Supplementary Fig. 4A). Moreover, the expression of several E2F factors was significantly downregulated in ND095 fibroblasts (Supplementary Fig. 4B). However, in ND120 we could not find enrichment of cell cycle related genes, nor genes bound by E2F transcription factors in downregulated genes or downregulated E2F factors. Changes in EHTM1 and EHMT2 expression in patients’ fibroblasts Interestingly, the expression of EHMT2 was significantly downregulated in ND095 compared to healthy control fibroblasts suggesting that p.Ala1077Ser has consequences for EHMT2 mRNA expression (Supplementary Fig. 5A). Despite this, the reads covering the NM_006709.5:c.3229G > T region showed a balance of 50% WT and 50% mutant transcripts (Supplementary Fig. 5B). In patient ND120, we detected lower mRNA expression of both EHMT1 and EHMT2 (Supplementary Fig. 5A). Reads in the EHMT1 NM_024757.5:c.1881delT region were predominantly WT suggesting that the T deletion affects the levels of mRNA likely by nonsense-mediated decay (Supplementary Fig. 5B). DISCUSSION We describe here a patient with a de novo pathogenic missense variant in the EHMT2 gene that causes a Kleefstra-like syndrome phenotype. This missense variant causes the change of alanine 1077 to serine in the catalytic SET domain of EHMT2 reducing its affinity for histone H3 tail and its catalytic activity by three- to five-fold. Interestingly, the presence of the p.Ala1077Ser change not only reduces the affinity of the catalytic domain for histone H3 but also abrogates the specificity of H3 recognition dependent on its modification. Loss of affinity of EHMT2 catalytic domain for the H3 tail once is dimethylated might favor the recruitment of other methyltransferases able to trimethylate histone H3. This recruitment might be blocked by the low but persistent binding of the p.Ala1077Ser mutant domain to H3K9me2. Although the changes in gene expression and H3K9 methylation detected in patients’ fibroblasts are more dramatic in ND095 than ND120, there is a significant correlation in changes of gene expression between both patients. In both cases, there is an upregulation of genes involved in the morphogenesis of diverse organs and tissues, suggesting that EHMT1/2 play a role in preventing the expression of genes belonging to alternative lineages. In accordance, EHMT1/2 have been previously involved in the silencing of alternative lineage genes during hematopoietic differentiation 34 , 35 . Regarding downregulated genes, a significant enrichment in cell cycle genes and E2F targets was only observed in patient ND095. Similarly, the depletion of EHTM2 in myoblasts has been previously shown to affect cell cycle through downregulation of E2F target genes 36 . This function has been suggested to be independent of EHMT2 methyltransferase activity and through its association with the E2F1/PCAF complex. Also, a downregulation of cell cycle genes and E2F-regulated factors has been described in KS induced pluripotent stem cells-derived neurons when compared to their healthy counterparts 37 . It is surprising that while patients with alterations in EHMT1 are known since 2004, reported as a syndrome caused by subtelomeric deletions of chromosome 9q 38 , individuals with genetic alterations in EHMT2 have not yet been described. In contrast, EHMT2 was reported to be a histone methyltransferase before EHMT1 6 , and has been more intensively studied. Both proteins are highly similar, widely expressed in human tissues and together with the H3K9 methylation mark have been described to play critical roles in differentiation and development. Despite this, several studies suggest that EHMT2 and EHMT1 might have different functions being EHMT2 more relevant for maintaining the cellular levels of H3K9 methylation 9 , 39 , 40 . Although both EHMT1 and EHMT2 can methylate histones in vitro on their own, heterodimers between both proteins are needed to display maximum methylation activity in vivo 9 . Dimer formation-competent but enzymatically inactive mutant Ehmt1 but not Ehmt2 can rescue specific knock out (KO) mouse embryonic stem cells (mESCs) from defective H3K9 methylation, suggesting that the catalytic activity of EHMT1 but not EHMT2 is dispensable for the complex H3K9 methyltransferase activity in vivo 39 . In addition, it has been suggested that some of the defects observed after EHMT1 depletion could be due to altered EHMT2 activity. The Ehmt1 KO in mESCs results in reduced Ehmt2 expression likely due to the impossibility to form heterodimers, while the Ehmt2 KO had no effects in Ehmt1 expression 9 , 40 . In addition, KS causing EHMT1 variants p.Cys1073Tyr and p.Arg1197Trp, which have been reported to have both defective in vitro activity and interaction with EHMT2, did not rescue the levels of H3K9 methylation nor restored EHMT2 levels when overexpressed in Ehmt1 KO mESCs 40 . Therefore, it is likely that the EHMT1 truncations observed in Kleefstra syndrome patients affect the catalytic capacity of EHMT2 through alterations in heterodimer formation. These observations agree with the reduced levels of EHMT2 expression found in the KS patient ND120. Our H3K9 methylation and gene expression data suggest that EHMT2 loss-of-function has more dramatic consequences regarding loss of H3K9 methylation and altered gene expression than loss of EHMT1 function, despite the fact that the p.Ala1077Ser change still retains some catalytic activity. This data agrees with the previously discussed more relevant role for EHMT2 in controlling the levels of H3K9 methylation than EHMT1. In this context, it is possible that loss of EHMT2 function might be more detrimental to cells than loss of EHMT1 function explaining why inactivating mutations in EHMT2 compatible with life are very rare. In agreement, mouse models have shown that the phenotypes of Ehmt1−/−embryos were mostly identical to those of Ehmt2−/− embryos, both leading to embryonic lethality 9 , 10 . Therefore, it is expected that complete loss of any of these proteins in humans results in severe developmental defects and are not compatible with life. In accordance with the human phenotype, both Ehmt1+/- and Ehmt2+/- mice are viable and recapitulate certain neurological traits observed in KS patients 41 . However, the interpretation of the effects of missense variants versus truncating variants is not straightforward. Indeed, according to GnomAD 42 V4 EHMT1 is much more intolerant to loss-of-function variants than EHMT2 (pLI = 1 and LEUOF = 0.1 90%(0.07–0.16) vs. pLI = 0.86 and LEUOF = 0.39 90%(0.31–0.49), respectively), while EHMT2 is more intolerant to missense variants (Z = 1.98 and o/e = 0.87 (0.84–0.91) for EHMT1 vs. Z = 4.63 and o/e = 0.68 90% (0.64–0.71) for EHMT2 ). While tools like DOMINO 43 , which predicts pathogenicity of genes based on their properties rather than variants, suggest a very likely dominant inheritance for EHMT2 in Mendelian disorders (probability: 0.8349), GnomAD probabilities suggest that monoallelic inactivating mutations could be tolerated. If this is the case, the pathogenicity of the monoallelic alteration in patient ND095 could be explained by potential dominant negative effects caused by the EHMT2 p.Ala1077Ser variant, such as sequestering EHMT1 into less active heterodimeric forms, that could lead to more severe effects than just the loss of one allele. Consequently, only a limited number of EHMT2 non-truncating variants could act via this mechanism which could also explain the rarity of cases. Importantly, a clear correlation between EHMT1 pathogenic variants and phenotype severity has not been yet established for KS 44 , reflecting the complexity of variant interpretation. In addition to deletions in chromosome 9q34.3 that eliminate one EHMT1 copy, most common forms of EHMT1 loss-of-function reported in KS patients are single nucleotide changes that cause frameshifts. Missense mutations are rarer and only a couple have been properly validated 40 . Larger efforts would be needed to evaluate the effects of EHMT1 and EHMT2 missense variants and their consequences for complex function and disease severity. Regarding diagnostic perspectives for patients with EHMT2 alterations, we found a DNA methylation signature for patient ND095 that resembles KS patients, likely due to the formation of a complex between EHMT1 and EHMT2. This shared, but slightly different, DNA methylation profile has been observed also for other disorders of the same protein complex such as BAFopathies 45 . However, the increased number of patients with rare diseases tested for DNA methylation profiles has improved the resolution and specificity of the episignatures ranging from altered protein complexes to genes, protein domains, and even single nucleotides 28 . Therefore, it is possible that a specific DNA methylation episignature for EHMT2 loss-of-function, different from that of patients with KS, can be established in the future after the identification and profiling of more patients with pathogenic EHMT2 alterations. Declarations Acknowledgements This study used tools provided through the RD-Connect GPAP, which received funding originally from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 305444. We thank the Bioinformatics Unit at ISCIII (I. Cuesta and S. Monzon) for their help in exome analysis, and A. Krepischi and L. Machado for discussions. We also thank the patients’ association Kleefstra España, the National Biobank of Rare Diseases (BioNER), the patients, their families, and their physicians, as well as the entire SpainUDP consortium. The authors appreciate the support of the Undiagnosed Diseases Network International (UDNI) for data sharing. Funding This work was funded with project PID2021-128087OB-I00 by MCIN /AEI /10.13039/501100011033 / FEDER, UE to M.J.B, and AESI PT20CIII/00009 (ISCIII Platform of Biobanks and Biomodels PT-20). Funding was also partially provided by the Genome Canada and the Ontario Genomics Institute Genomics Applications Partnership Program Grant (OGI-188). Author Contribution Statement BMD and MJB conceived and designed the work. BMD, ELM, JG, EBS, MP, PMR, RCC, DR, TK, JK, JR, BS and MJB participated in data collection. JK, JR, BS and MJB participate in data analysis. PMR, RCC, LLJ, LMM, MFP, EHS, GGM, BB and DSP generated critical reagents for the study. ABN, MHM and MJB carried out functional assays. MJB coordinated the research and wrote the manuscript with input from all authors. All authors approved the final version of the manuscript. Ethical Approval Informed consents were signed by patient’s legal representatives. This research project was approved by the ISCIII Research Ethics Committee entry number CEI PI 03_2022. Competing Interests Authors declare no competing interest Data availability RNA-seq raw data is available upon request to the corresponding author. The pathogenic EHMT2 NM_006709.5:c.3229G > T variant detected in our study has been submitted to ClinVar ( https://www.ncbi.nlm.nih.gov/clinvar/ , submission number: SUB14159160). References Kleefstra T, Brunner HG, Amiel J et al. Loss-of-Function Mutations in Euchromatin Histone Methyl Transferase 1 (EHMT1) Cause the 9q34 Subtelomeric Deletion Syndrome. 2006www.ajhg.org. Willemsen MH, Vulto-Van Silfhout AT, Nillesen WM et al. Update on Kleefstra syndrome. Mol Syndromol 2012; 2 : 202–212. Kleefstra T, Kramer JM, Neveling K et al. Disruption of an EHMT1-associated chromatin-modification module causes intellectual disability. Am J Hum Genet 2012; 91 : 73–82. Faundes V, Newman WG, Bernardini L et al. 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G9a promotes proliferation and inhibits cell cycle exit during myogenic differentiation. Nucleic Acids Res 2016; 44 : 8129–8143. Fear VS, Forbes CA, Anderson D et al. CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis: pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency. Stem Cell Res Ther 2022; 13 . doi:10.1186/s13287-022-02740-3. Harada N, Visser R, Dawson A et al. A 1-Mb critical region in six patients with 9q34.3 terminal deletion syndrome. J Hum Genet 2004; 49 : 440–444. Tachibana M, Matsumura Y, Fukuda M, Kimura H, Shinkai Y. G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO J 2008; 27 : 2681–2690. Yamada A, Shimura C, Shinkai Y. Biochemical validation of EHMT1 missense mutations in Kleefstra syndrome. J Hum Genet 2018; 63 : 555–562. Balemans MCM, Huibers MMH, Eikelenboom NWD et al. Reduced exploration, increased anxiety, and altered social behavior: Autistic-like features of euchromatin histone methyltransferase 1 heterozygous knockout mice. Behavioural Brain Research 2010; 208 : 47–55. Karczewski KJ, Francioli LC, Tiao G et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 2020; 581 : 434–443. Quinodoz M, Royer-Bertrand B, Cisarova K, Di Gioia SA, Superti-Furga A, Rivolta C. DOMINO: Using Machine Learning to Predict Genes Associated with Dominant Disorders. The American Journal of Human Genetics 2017; 101 : 623–629. Ciaccio C, Scuvera G, Tucci A et al. New Insights into Kleefstra Syndrome: Report of Two Novel Cases with Previously Unreported Features and Literature Review. Cytogenet Genome Res 2018; 156 : 127–133. Aref-Eshghi E, Bend EG, Hood RL et al. BAFopathies’ DNA methylation epi-signatures demonstrate diagnostic utility and functional continuum of Coffin–Siris and Nicolaides–Baraitser syndromes. Nat Commun 2018; 9 : 4885. Kleefstra T, van Zelst-Stams WA, Nillesen WM et al. Further clinical and molecular delineation of the 9q subtelomeric deletion syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype. J Med Genet 2009; 46 : 598–606. Additional Declarations There is no duality of interest Supplementary Files Supplementarytextandfigures.pdf Supplementarytable1.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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A. Patient pictures showing dimorphisms. B. Confirmation by Sanger sequencing of variant NM_006709.5:c.3229G\u0026gt;T (hg19 chr6:31848838C\u0026gt;A) in the proband ND095 and the absence of variant in both parents. C. p.Ala1077 is located in the catalytic domain of EHMT2. D. Protein alignment of several human histone methyltranserases. p.Ala1077 and other amino acids described to confer gain of function are highlighted. Adapted from Kato S. et al. E. Structure of EHMT2 SET-domain dimers with histone H3 (purple) and SAM (blue). F. Magnification shows that residue p.Ala1077 in EHMT2 stablishes hydrogen bonds (highlighted in orange) with histone tail residue Thr6 and EHMT2 residue p.Arg1088.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3893528/v1/29a041816d808e7d6292ff9f.png"},{"id":51136160,"identity":"1838b0fc-3ffd-4bb3-8b33-ead4c8e83113","added_by":"auto","created_at":"2024-02-14 18:34:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":277755,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular alterations in EHMT2 catalytic domain caused by the p.Ala1077Ser change. A. Measure of cumulative methyltransferase activity over time (seconds) using 250 ng of the catalytic domain of EHMT2 wild type (WT) or p.Ala1077Ser mutant and 2.5 µg of recombinant histone H3 as a substrate. Graph shows the mean and standard deviation of two replicates. B. Specific activity in nmol/min/mg of WT and p.Ala1077Ser catalytic domains. Mean and standard deviation of two replicates is shown. C. Coomassie blue stained gel depicting the interaction of WT and Ala1077 catalytic domains with histone H3 peptides amino acids 1 to 21 unmodified (H3), monomethylated at lysine 9 (H3k9me1) and dimethylated at lysine 9 (H3k9me3). D. Quantification of WT and p.Ala1077Ser catalytic domains binding to peptides relative to input. Mean and standard deviation of three quantifications is shown. P-values for comparisons between WT and p.Ala1077Ser correspond to *\u0026lt;0.05, **\u0026lt;0.005 and ***\u0026lt;0.0005\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3893528/v1/87315a2273d4a450f6b7ca8e.png"},{"id":51136163,"identity":"80856386-3a07-4c17-a723-a81ec6e003d0","added_by":"auto","created_at":"2024-02-14 18:34:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1669470,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the Episign test in patient ND095 A. Hierarchical clustering and B. multidimensional scaling plots indicate the case (red) has a DNA methylation profile similar to subjects with a confirmed Kleefstra episignature (blue) and distinct from controls (green). C. MVP score, a multi-class supervised classification system capable of discerning between multiple episignatures by generating a probability score for each episignature. The elevated score for Kleefstra syndrome indicates an episignature similar to the Kleefstra syndrome reference.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3893528/v1/7c19d297d4c08b2b03820cfe.png"},{"id":51136161,"identity":"f2a93385-c63d-42a0-8ddd-088089d0f270","added_by":"auto","created_at":"2024-02-14 18:34:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1507690,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic and epigenetic analysis of fibroblasts derived from a healthy control, a Kleefstra Syndrome patient (ND120) and the patient carrying the EHMT2 p.Ala1077Ser variant (ND095). A. Relative abundance of histone H3 modifications quantified by mass spectrometry in patient's fibroblasts and a healthy donor. Bars show the mean and standard deviation of tripicates. Significant differences compared to control healthy fibroblasts are indicated with asterisks according to P-values *\u0026lt;0.05, **\u0026lt;0.005 and ***\u0026lt;0.0005. B. Overlap of upregulated and downregulated genes found in ND095 or ND120 compared to the healthy control at adjusted p-value\u0026lt;0.05. C. Scatter plot showing correlation between expression fold change of all genes in ND120 or ND095 compared to control fibroblasts. Pearson's correlation coefficient is shown. Genes changing expression at an adjusted p-value\u0026lt;0.05 only in ND120 (pink), only in ND095 (purple), in both (orange) and not significantly changing expression (green) are shown. D. Network representation and clustering of GSEA results on GOBP terms at FDR\u0026lt;0.06. Red nodes correspond to terms enriched in genes upregulated in ND095 compared to control fibroblasts and blue nodes in downregulated genes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3893528/v1/8b33bc083a4a058c5b5a502a.png"},{"id":71667150,"identity":"1dd336ae-f0ca-44b6-b7e1-e5ba9925abb4","added_by":"auto","created_at":"2024-12-17 14:36:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7874141,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3893528/v1/ff771cbf-4f96-40b7-b962-708d44ac4d8a.pdf"},{"id":51136162,"identity":"34e13453-9206-4720-ab82-47518e550cdb","added_by":"auto","created_at":"2024-02-14 18:34:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1331420,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytextandfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3893528/v1/ada9a53a6a5e7e7d6427a667.pdf"},{"id":51136165,"identity":"4271d63d-88bd-466c-8d4c-0c15d7ab48bc","added_by":"auto","created_at":"2024-02-14 18:34:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7325748,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3893528/v1/0a9fbdbf7265924592c001e9.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"\u003cp\u003eEhmt2 Loss-of-function Alterations Cause a Kleefstra-like Syndrome\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDysregulation of the epigenetic machinery is associated with neurodevelopmental defects in humans. Kleefstra syndrome (KS) is a neurodevelopmental syndrome caused by heterozygous deletions at chromosome 9q34.3 that include the \u003cem\u003eEHMT1\u003c/em\u003e gene (~\u0026thinsp;50%) or heterozygous intragenic \u003cem\u003eEHMT1\u003c/em\u003e pathogenic variants (~\u0026thinsp;50%)\u003csup\u003e1,2\u003c/sup\u003e. In addition, loss-of-function mutations in the \u003cem\u003eKMT2C\u003c/em\u003e (MLL3) gene cause a Kleefstra-like syndrome called Kleefstra syndrome-2 (KLEFS2)\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Moreover, although very rare, de novo mutations in epigenetic regulators genes \u003cem\u003eMBD5\u003c/em\u003e, \u003cem\u003eMLL3\u003c/em\u003e, \u003cem\u003eSMARCB1\u003c/em\u003e, and \u003cem\u003eNR1I3\u003c/em\u003e have been associated with intellectual disability disorders that fall into the Kleefstra syndrome\u0026rsquo;s phenotypic spectrum\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEHMT1 (also known as GLP) and EHMT2 (also known as G9a) are highly homologous proteins that contain a SET domain that confers them the ability to mono- and dimethylate lysine 9 at histone H3 (H3K9)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. H3K9 methylation is finely regulated by several methyltransferases and demethylases and plays important roles in development, differentiation, and cancer\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. EHMT1 and EHMT2 can form homo- or heterodimers through SET domain interactions, being heterodimers the most active configuration and the preferred form in cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Both Ehmt1-/- and Ehmt2-/- mice show severe developmental delays and early embryonic lethality that correlates with lower levels of H3k9me1 and me2\u003csup\u003e9,10\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the overlap in domains and functions of EHMT1 and EHMT2 and their relevance for development, patients with alterations in EHMT2 have never been reported. Here we report a Kleefstra-like syndrome case with a missense variant in the catalytic SET domain of EHMT2 that causes alanine 1077 change to serine reducing its affinity for the H3 tail and the activity of the enzyme by three- to five-fold. Results from our study allow defining this variant as pathogenic following the ACMG criteria\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, since it appears the novo in a patient with disease and no family history (PS2), in vitro functional studies are supportive of a damaging effect (PS3), it is absent from controls in GnomAD (PM2) and the variant is located in a critical functional domain (PM1).\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003ePatients ND095 and ND120 were recruited by the undiagnosed rare diseases program SpainUDP\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e at the Institute of Rare Diseases Research (IIER), Spanish National Institute of Health Carlos III (ISCIII). Peripheral blood samples and skin biopsies were collected from patients and their parents to perform trio-based whole-exome sequencing and establishment of fibroblast cultures respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWhole Exome sequencing\u003c/h2\u003e \u003cp\u003eWhole exome sequencing and data analysis was performed on the probands and their unaffected parents. Genomic DNA was extracted from peripheral blood using the Qiagen QIAamp DNA kit. Whole Exome Sequencing (WES) libraries were prepared using the Nimblegen MedExome\u0026thinsp;+\u0026thinsp;ChrMit as enrichment kit and HS2000 v4, 2x100bp sequencing platform in the ND095 family and Nextera Felx DNA Library Prep and Illumina NextSeq500 in the ND120 family. Data analysis was performed using two different standardized protocols as previously described\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These included an in-house analysis and a parallel analysis using the Genomic Analysis module of the RD-Connect Genome-Phenome Analysis Platform (GPAP)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. All commonly identified rare variants were further analysed, checking all available scientific evidence through detailed searches in public databases (including Gene-Card, NCBI, UniProt, OMIM, PubMed and ExAc). Sanger sequencing was performed to validate candidate variants using the following primers for EHMT2 5\u0026acute;-AGCAGGGTAAGGAGGGTCTC-3\u0026rsquo; and 5\u0026acute;-CCACCTCCTAATAGCCCACA-3\u0026rsquo;, and for EHMT1 5\u0026rsquo;-CTTCTTCTCTGTGGGGCGAG-3\u0026acute; and 5\u0026acute;-CACCATAAGCATCAGCATCAGC-3\u0026acute;\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eFibroblast culture\u003c/h2\u003e \u003cp\u003eHuman dermal fibroblasts cultures were established from skin tissue samples. Briefly, fibroblasts were mechanically isolated by dissecting the dermal layer of the skin and the resulting fragments were incubated at 37\u0026deg;C in Dulbeccos' modified Eagle's medium (DMEM) containing 2% of fetal calf serum. Cells were expanded by incubation in 75 cm2 culture flasks at 37\u0026deg;C with 5% CO2 and 95% humidity in DMEM containing 10% fetal bovine serum.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from fibroblasts using the RNeasy mini kit from Qiagen. RNA-seq was performed at BGI Tech Solutions with two or three biological replicates per condition. Briefly, ribosomal RNAs were removed using a RNase H-based method and first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP, end repair, A addition and adaptor ligation. The U-labeled second-strand template was digested with Uracil-DNA-Glycosylase (UDG) and amplified by PCR. The resulting library was validated by quality control. The PCR products were then heat denatured and circularized by the splint oligo sequence to generate a single strand circle DNA followed by rolling circle replication to create DNA nanoballs (DNB) for sequencing on the MGI DNBSEQ platform. Raw sequencing data with adapter sequences or low-quality sequences was trimmed or filtered and examined by FastQC for basic quality controls. The sequencing analysis was carried out using Galaxy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://usegalaxy.eu/\u003c/span\u003e\u003cspan address=\"https://usegalaxy.eu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Paired reads were aligned to the human hg19 genome build using STAR\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Gene counts were calculated using HTseq-count\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and differential expression between patients and healthy fibroblast was interrogated using DEseq\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Differentially expressed genes were considered at an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eFor enrichment analysis in differentially expressed genes, we used GSEA preranked\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e with genes ranked according to p-value corrected log2 of fold change. Interrogated gene sets were Gene Ontology Biological Process (GOBP) and transcription factor targets (TFT). In addition, we performed enrichment in GOBP terms in significantly upregulated or downregulated genes using Panther (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pantherdb.org/\u003c/span\u003e\u003cspan address=\"https://pantherdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e18\u003c/sup\u003e. Scatterplots showing correlations were generated with ggplot2 in Galaxy. Pearson\u0026rsquo;s correlation and bubble plots were calculated using SRPlot\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Network representation of enriched GOBP terms was generated using Cytoscape Enrichment Map and AutoAnotate Apps\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProduction of recombinant proteins\u003c/h2\u003e \u003cp\u003epET28a-LIC containing the catalytic domain of human EHMT2 (aa913-1193) was a gift from Cheryl Arrowsmith (Addgene plasmid # 25503; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:25503\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:25503\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:Addgene_25503). Mutation p.Ala1077Ser was introduced using the QuikChange II XL Site-Directed Mutagenesis Kit from Agilent. Expression and purification of His-tagged EHMT2 catalytic domain WT and p.Ala1077Ser in bacteria has been previously described\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Recombinant H3 was expressed in \u003cem\u003eE. coli\u003c/em\u003e and purified as previously described\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistone methyltransferase assays\u003c/h2\u003e \u003cp\u003eHistone methyltransferase assays were performed using the fluorescence-based assay SAM Methyltransferase Assay SAMfluoro\u0026trade; from G-Biosciences that detects the formation of resorufin over time as a result of the methyltransferase reaction. Assays were performed using 250, 500 or 1000 ng of His-tagged WT or p.Ala1077Ser EHMT2 catalytic domains and 2.5 \u0026micro;g of recombinant histone H3 following the manufacturer instructions. Cumulative reads were measured every minute for 45 minutes. Specific activity was calculated using a resorufin standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistone peptide binding assays\u003c/h2\u003e \u003cp\u003e10 \u0026micro;g of biotinylated histone 3 peptides aa 1 to 21 (Active Motif) unmodified, monomethylated at K9 or dimethylated at K9 were incubated overnight at 4\u0026ordm;C with 5 ug of recombinant EHMT2 wild type or p.Ala1077Ser mutant catalytic domain in 300 \u0026micro;l of binding buffer 50 mM Tris pH 7.5, 150 mM NaCl, 0.05% NP-40 and 1 mM PMSF. After 1 hour incubation with Pierce streptavidin magnetic beads and extensive washing with binding buffer bound proteins were resolved by SDS-PAGE and stained with Coomassie blue. Gels were scanned using the Bio-Rad GelDoc Go Imaging System and bands quantified using ImageJ\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eProtein modeling\u003c/h2\u003e \u003cp\u003ePDB Protein Data Bank entry 5JJ0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2210/pdb5JJ0/pdb\u003c/span\u003e\u003cspan address=\"10.2210/pdb5JJ0/pdb\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) corresponding to the catalytic domain of EHMT2 complexed with the histone peptide H3K9M and SAM was explored in 3D using PDB resources\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEpiSign assay\u003c/h2\u003e \u003cp\u003eMethylation analysis was conducted using the clinically validated EpiSign assay, following previously established methods\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Methylated and unmethylated signal intensities generated from the EPIC array were imported into R 3.5.1 for normalization, background correction, and filtering. Beta values were then calculated as a measure of methylation level, ranging from 0 (no methylation) to 1 (complete methylation), and processed through the established support vector machine (SVM) classification algorithm for EpiSign disorders. The classifier utilized the EpiSign Knowledge Database, which consists of over 10,000 methylation profiles from reference disorder-specific and unaffected control cohorts, to generate disorder-specific methylation variant pathogenicity (MVP) scores. These MVP scores are a measure of prediction confidence for each disorder and range from 0 (discordant) to 1 (highly concordant). A positive classification typically generates MVP scores greater than 0.5. The final matched EpiSign result is generated using these scores, along with the assessment of hierarchical clustering and multidimensional scaling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetection of histone modifications by mass spectrometry\u003c/h2\u003e \u003cp\u003eMass spectrometry was performed by Active Motif. Histones were acid extracted from a cell pellet containing 2.5x10\u003csup\u003e6\u003c/sup\u003e cells, derivatized via propionylation, digested with trypsin, newly formed N-termini were propionylated as previously described\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Histones were extracted by incubating samples at room temperature for 1 hour in 0.2M sulfuric acid with intermittent vortexing. Histones were then precipitated by the addition of trichloroacetic acid (TCA) on ice, and recovered by centrifugation at 10,000 x g for 5 minutes at 4\u0026deg;C. The pellet was then washed once with 1mL cold acetone/0.1% HCl and twice with 100% acetone, and then air dried in a clean hood. The histones were propionylated by adding 1:3 v/v propionic anhydride/2-propanol and incrementally adding ammonium hydroxide to keep the pH around 8, and subsequently dried in a SpeedVac concentrator. The pellet was then resuspended in 100 mM ammonium bicarbonate and adjusted to pH 7\u0026ndash;8 with ammonium hydroxide. The histones were then digested with trypsin, resuspended in 100mM ammonium bicarbonate overnight at 37\u0026deg;C, and dried in a SpeedVac concentrator. The pellet was resuspended in 100mM ammonium bicarbonate and propionylated a second time by adding 1:3 v/v propionic anhydride/2-propanol and incrementally adding ammonium hydroxide to keep the pH around 8, and subsequently dried in a SpeedVac concentrator. Histone peptides were resuspended in 50 \u0026micro;L of 0.1% TFA and 3 \u0026micro;l were injected with 3 technical replicates in a Thermo Scientific TSQ Quantum Ultra mass spectrometer coupled with an UltiMate 3000 Dionex nano-liquid chromatography system. The data was quantified using Skyline\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and represents the percent of each modification within the total pool of that amino acid residue.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical characteristics of the proband overlap with Kleefstra syndrome\u003c/h2\u003e \u003cp\u003eProband ND095 was remitted to the undiagnosed rare diseases program SpainUDP\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e at the age of 4.8 years. Despite exhaustive clinical and genetic studies, mainly through candidate gene panels, the patient had remained undiagnosed until that time. The proband is a male that suffered from intrauterine growth restriction and congenital anomalies, including coarctation of the aorta and renal medullar cystic disease. At the age of 2 years, he was diagnosed with global developmental delay, characterized by slow developing of gross motor milestones and expressive language delay, hypotonia without weakness and nephrocalcinosis. At the craniofacial level, he had brachycephaly, plagiocephaly with very flat occiput, broad face and midface hypoplasia, synophrys, sparse medial eyebrows, epicanthus, lateral deviation of upslanted palpebral fissures, mildly everted lower eyelids, palpebral ptosis (right eye), anteverted nares, smooth philtrum and carp-like mouth. In addition, he had dental diastema, microdontia, persistent fetal fingertip pads and scoliosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary File 1). From the clinical point of view, the patient\u0026rsquo;s phenotype overlaps significantly with the clinical characteristics of KS (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnomalies found in patient ND095 compared to Kleefstra syndrome\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnomalies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND095\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKleefstra\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh birth weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u0026ndash;50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicrocephaly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026ndash;80%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrachycephaly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlat face\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMidface hypoplasia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55\u0026ndash;100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypertelorism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55\u0026ndash;70%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArched eyebrows\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSynophrys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55\u0026ndash;80%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUpslanting palpebral fissures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026ndash;70%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpicanthal folds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026ndash;70%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOcular anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnteverted nares\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026ndash;80%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarp mouth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtruding tongue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026ndash;60%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDental anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;15%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEar anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45\u0026ndash;80%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypoacusia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;30%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCardiovascular anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026ndash;45%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUmbilical/inguinal hernia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u0026ndash;20%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal issues\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u0026ndash;30%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenital anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45\u0026ndash;50% of males\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkeletal anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026ndash;50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimb anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrachydactyly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShort stature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;39%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eObesity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026ndash;40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypotonia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u0026ndash;80%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsychomotor delay/intellectual disability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpeech anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enon-verbal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e~\u0026thinsp;100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAutism ASD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026ndash;75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBehavioral problems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003estereotypy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65\u0026ndash;70%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSleep disorder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrain imaging anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epartial empty sella turcica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u0026ndash;60%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpilepsy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eone episode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGastro-esophageal reflux\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u0026ndash;19%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTracheomalacia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;11%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRespiratory complications\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;14%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecurrent infections\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u0026ndash;64%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWhole-exome sequencing revealed a variant of uncertain significance (VUS) in the\u003c/b\u003e \u003cb\u003eEHMT2\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhole-exome sequencing of patient ND095 and his parents, carried out at SpainUDP, revealed no detectable deleterious variants in the KS causing gene \u003cem\u003eEHMT1\u003c/em\u003e. In addition, no alterations were detected in \u003cem\u003eKMT2C\u003c/em\u003e, which causes Kleefstra syndrome 2\u003csup\u003e5\u003c/sup\u003e. Similarly, no pathogenic variants were detected in chromatin-related genes \u003cem\u003eMBD5\u003c/em\u003e, \u003cem\u003eMLL3\u003c/em\u003e, \u003cem\u003eSMARCB1\u003c/em\u003e, and \u003cem\u003eNR1I3\u003c/em\u003e, which have been previously associated with Kleefstra-like syndromes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Surprisingly, the sequencing revealed a heterozygous de novo variant in the \u003cem\u003eEHMT2\u003c/em\u003e gene (hg19 chr6:31848838C\u0026thinsp;\u0026gt;\u0026thinsp;A; NM_006709.5:c.3229G\u0026thinsp;\u0026gt;\u0026thinsp;T) that resulted in the amino acid change alanine 1077 to serine (p.Ala1077Ser) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This variant was not present in GnomAD V4 genomes nor exomes, and neither was it reported in ClinVar. Several in silico predictors classified this variant as pathogenic (EIGEN, FATHMM, LRT, MutPred MVP, REVEL) while it was classified as a VUS by others (Mutation Taster, Mutation Assessor, SIFT, PROVEAN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe p.Ala1077Ser variant decreases the activity of EHMT2\u003c/h2\u003e \u003cp\u003eAla1077 is located in the catalytic SET domain of EHMT2, in a highly conserved region among human histone methyltransferases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Ala1077 is, however, far from the AdoMet cofactor interacting amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and the presumed dimerization region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The published EHMT2 catalytic domain structure in complex with H3 tail\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e shows that Ala1077 interacts with the H3 tail residue threonine 6, suggesting that it might affect the catalytic activity of EHMT2 through changes in this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To test this possibility, we first evaluated the effects of the p.Ala1077Ser variant in the activity of the enzyme. We used an in vitro fluorescence-based assay that allows the detection of methyltransferase activity over time to test the activity of both wild type and Ala1077Ser EHMT2 catalytic domains expressed and purified in \u003cem\u003eE. coli\u003c/em\u003e, on recombinant histone H3. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows that the p.Ala1077Ser variant drastically reduces the methyltransferase activity of EHMT2 on histone H3 over time and the specific activity of the enzyme by five-fold, respectively. Replication of the histone methyltransferase assay using different EHMT2 concentrations shows that the Ala1077Ser variant reduces the specific activity of the enzyme by three- to five-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;2). Next, we evaluated if the p.Ala1077Ser variant affects the interaction of EHMT2 with the histone H3 tail by testing the interaction of both EHMT2 wild type and the p.Ala1077Ser mutant with synthetic H3 peptides (aa 1\u0026ndash;21), either unmethylated, mono or dimethylated at lysine 9, in an in vitro pull-down assay. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD show that the p.Ala1077Ser variant significantly reduces de affinity of the catalytic domain for the histone H3 tail. In addition, we detected a reduced affinity of the wild type catalytic domain for the dimethylated H3 peptide compared to unmethylated or monomethylated peptides. These results are in line with previous data that suggest a loss of affinity of methyltransferases for their substrate once it is fully methylated\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This behavior was not observed in the mutant catalytic domain (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Episign test classifies patient ND095 as Kleefstra syndrome.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to investigate the overlap of patient ND095 with KS specific DNA methylation signature, we compared the blood DNA methylation profile of patient ND095 with Kleefstra patients using version three of the test EpiSign\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This test, based in blood DNA methylation profiles, allows the diagnosis of over fifty different genetic diseases. Unsupervised hierarchical clustering of DNA methylation profiles shows that ND095 clusters with Kleefstra syndrome patients and clearly separates from healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The computational model classified patient ND095 as KS with high confidence, generating a low score for other 56 syndromes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, the principal component analysis of DNA methylation showed that ND095 had a particular methylation profile that differs slightly from KS patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This data suggests the involvement of an \u003cem\u003eEHMT1\u003c/em\u003e-related but different causal gene in the phenotype of patient ND095.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOverlap of molecular signatures between patient ND095 and Kleefstra syndrome.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we established fibroblasts cultures from a healthy donor, the ND095 patient carrying the EHMT2 p.Ala1077Ser variant and a patient (ND120) recently diagnosed with KS through our program, who carries a frameshift variant in \u003cem\u003eEHMT1\u003c/em\u003e (NM_024757.5:c.1881delT p.His620ThrfsTer12), and quantified histone modifications by mass spectrometry. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows that ND095 fibroblasts had decreased levels of mono-, di- and trimethylated H3K9, while levels of H3K27me3 remained unchanged. In correlation, we found an increase in unmodified and acetylated H3K9. Similar but less dramatic effects were found in KS patient ND120 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These results are in agreement with a previous characterization of changes in histone modifications after knockdown of both \u003cem\u003eEHMT2\u003c/em\u003e and \u003cem\u003eEHMT1\u003c/em\u003e\u003csup\u003e33\u003c/sup\u003e and are compatible with the loss of EHMT2 activity in patient ND095.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfterwards, we carried out RNA-seq in the fibroblasts cultures and identified differentially expressed genes (DEGs) in ND095 and ND120 fibroblasts compared to healthy control fibroblasts (Supplementary Table\u0026nbsp;1). Among DEGs we found both upregulated and downregulated genes, with a higher number of DEGs in ND095 compared to ND120 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This correlates with the more severe effects on H3K9 methylation in ND095 fibroblasts found by mass spectrometry. Despite this difference, there was a significant overlap in down and upregulated genes between ND095 and ND120 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and a significant correlation comparing the fold change of expression of all genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWe next investigated the enrichment of GOBP terms in the DEGs using gene set enrichment analysis (GSEA). Results show a significant enrichment of genes involved in the morphogenesis of diverse tissues and organs derived from the three embryonic layers, and genes involved in the extracellular matrix in genes upregulated in ND095 compared to control fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Significant enrichment in morphogenesis genes in upregulated genes was also identified in patient ND120 (Supplementary Fig.\u0026nbsp;3A) and in genes commonly upregulated in both patients (Supplementary Fig.\u0026nbsp;3B). Genes downregulated in ND095 fibroblasts compared to healthy control fibroblasts were mainly involved in cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Additionally, a significant enrichment of E2F transcription factors binding sites was found around the transcriptional start site of genes downregulated in ND095 fibroblasts (Supplementary Fig.\u0026nbsp;4A). Moreover, the expression of several E2F factors was significantly downregulated in ND095 fibroblasts (Supplementary Fig.\u0026nbsp;4B). However, in ND120 we could not find enrichment of cell cycle related genes, nor genes bound by E2F transcription factors in downregulated genes or downregulated E2F factors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in\u003c/b\u003e \u003cb\u003eEHTM1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eEHMT2\u003c/b\u003e \u003cb\u003eexpression in patients\u0026rsquo; fibroblasts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eInterestingly, the expression of \u003cem\u003eEHMT2\u003c/em\u003e was significantly downregulated in ND095 compared to healthy control fibroblasts suggesting that p.Ala1077Ser has consequences for \u003cem\u003eEHMT2\u003c/em\u003e mRNA expression (Supplementary Fig.\u0026nbsp;5A). Despite this, the reads covering the NM_006709.5:c.3229G\u0026thinsp;\u0026gt;\u0026thinsp;T region showed a balance of 50% WT and 50% mutant transcripts (Supplementary Fig.\u0026nbsp;5B). In patient ND120, we detected lower mRNA expression of both \u003cem\u003eEHMT1\u003c/em\u003e and \u003cem\u003eEHMT2\u003c/em\u003e (Supplementary Fig.\u0026nbsp;5A). Reads in the \u003cem\u003eEHMT1\u003c/em\u003e NM_024757.5:c.1881delT region were predominantly WT suggesting that the T deletion affects the levels of mRNA likely by nonsense-mediated decay (Supplementary Fig.\u0026nbsp;5B).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe describe here a patient with a de novo pathogenic missense variant in the \u003cem\u003eEHMT2\u003c/em\u003e gene that causes a Kleefstra-like syndrome phenotype. This missense variant causes the change of alanine 1077 to serine in the catalytic SET domain of EHMT2 reducing its affinity for histone H3 tail and its catalytic activity by three- to five-fold. Interestingly, the presence of the p.Ala1077Ser change not only reduces the affinity of the catalytic domain for histone H3 but also abrogates the specificity of H3 recognition dependent on its modification. Loss of affinity of EHMT2 catalytic domain for the H3 tail once is dimethylated might favor the recruitment of other methyltransferases able to trimethylate histone H3. This recruitment might be blocked by the low but persistent binding of the p.Ala1077Ser mutant domain to H3K9me2.\u003c/p\u003e \u003cp\u003eAlthough the changes in gene expression and H3K9 methylation detected in patients’ fibroblasts are more dramatic in ND095 than ND120, there is a significant correlation in changes of gene expression between both patients. In both cases, there is an upregulation of genes involved in the morphogenesis of diverse organs and tissues, suggesting that EHMT1/2 play a role in preventing the expression of genes belonging to alternative lineages. In accordance, EHMT1/2 have been previously involved in the silencing of alternative lineage genes during hematopoietic differentiation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Regarding downregulated genes, a significant enrichment in cell cycle genes and E2F targets was only observed in patient ND095. Similarly, the depletion of EHTM2 in myoblasts has been previously shown to affect cell cycle through downregulation of E2F target genes\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This function has been suggested to be independent of EHMT2 methyltransferase activity and through its association with the E2F1/PCAF complex. Also, a downregulation of cell cycle genes and E2F-regulated factors has been described in KS induced pluripotent stem cells-derived neurons when compared to their healthy counterparts\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is surprising that while patients with alterations in \u003cem\u003eEHMT1\u003c/em\u003e are known since 2004, reported as a syndrome caused by subtelomeric deletions of chromosome 9q\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, individuals with genetic alterations in \u003cem\u003eEHMT2\u003c/em\u003e have not yet been described. In contrast, EHMT2 was reported to be a histone methyltransferase before EHMT1\u003csup\u003e6\u003c/sup\u003e, and has been more intensively studied. Both proteins are highly similar, widely expressed in human tissues and together with the H3K9 methylation mark have been described to play critical roles in differentiation and development. Despite this, several studies suggest that EHMT2 and EHMT1 might have different functions being EHMT2 more relevant for maintaining the cellular levels of H3K9 methylation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Although both EHMT1 and EHMT2 can methylate histones in vitro on their own, heterodimers between both proteins are needed to display maximum methylation activity in vivo\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Dimer formation-competent but enzymatically inactive mutant Ehmt1 but not Ehmt2 can rescue specific knock out (KO) mouse embryonic stem cells (mESCs) from defective H3K9 methylation, suggesting that the catalytic activity of EHMT1 but not EHMT2 is dispensable for the complex H3K9 methyltransferase activity in vivo\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In addition, it has been suggested that some of the defects observed after EHMT1 depletion could be due to altered EHMT2 activity. The Ehmt1 KO in mESCs results in reduced Ehmt2 expression likely due to the impossibility to form heterodimers, while the Ehmt2 KO had no effects in Ehmt1 expression\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In addition, KS causing EHMT1 variants p.Cys1073Tyr and p.Arg1197Trp, which have been reported to have both defective in vitro activity and interaction with EHMT2, did not rescue the levels of H3K9 methylation nor restored EHMT2 levels when overexpressed in Ehmt1 KO mESCs\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, it is likely that the EHMT1 truncations observed in Kleefstra syndrome patients affect the catalytic capacity of EHMT2 through alterations in heterodimer formation. These observations agree with the reduced levels of EHMT2 expression found in the KS patient ND120.\u003c/p\u003e \u003cp\u003eOur H3K9 methylation and gene expression data suggest that EHMT2 loss-of-function has more dramatic consequences regarding loss of H3K9 methylation and altered gene expression than loss of EHMT1 function, despite the fact that the p.Ala1077Ser change still retains some catalytic activity. This data agrees with the previously discussed more relevant role for EHMT2 in controlling the levels of H3K9 methylation than EHMT1. In this context, it is possible that loss of EHMT2 function might be more detrimental to cells than loss of EHMT1 function explaining why inactivating mutations in EHMT2 compatible with life are very rare. In agreement, mouse models have shown that the phenotypes of Ehmt1−/−embryos were mostly identical to those of Ehmt2−/− embryos, both leading to embryonic lethality\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, it is expected that complete loss of any of these proteins in humans results in severe developmental defects and are not compatible with life. In accordance with the human phenotype, both Ehmt1+/- and Ehmt2+/- mice are viable and recapitulate certain neurological traits observed in KS patients\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, the interpretation of the effects of missense variants versus truncating variants is not straightforward. Indeed, according to GnomAD\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e V4 \u003cem\u003eEHMT1\u003c/em\u003e is much more intolerant to loss-of-function variants than \u003cem\u003eEHMT2\u003c/em\u003e (pLI = 1 and LEUOF = 0.1 90%(0.07–0.16) vs. pLI = 0.86 and LEUOF = 0.39 90%(0.31–0.49), respectively), while EHMT2 is more intolerant to missense variants (Z = 1.98 and o/e = 0.87 (0.84–0.91) for \u003cem\u003eEHMT1\u003c/em\u003e vs. Z = 4.63 and o/e = 0.68 90% (0.64–0.71) for \u003cem\u003eEHMT2\u003c/em\u003e). While tools like DOMINO\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, which predicts pathogenicity of genes based on their properties rather than variants, suggest a very likely dominant inheritance for \u003cem\u003eEHMT2\u003c/em\u003e in Mendelian disorders (probability: 0.8349), GnomAD probabilities suggest that monoallelic inactivating mutations could be tolerated. If this is the case, the pathogenicity of the monoallelic alteration in patient ND095 could be explained by potential dominant negative effects caused by the EHMT2 p.Ala1077Ser variant, such as sequestering EHMT1 into less active heterodimeric forms, that could lead to more severe effects than just the loss of one allele. Consequently, only a limited number of \u003cem\u003eEHMT2\u003c/em\u003e non-truncating variants could act via this mechanism which could also explain the rarity of cases. Importantly, a clear correlation between \u003cem\u003eEHMT1\u003c/em\u003e pathogenic variants and phenotype severity has not been yet established for KS\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, reflecting the complexity of variant interpretation. In addition to deletions in chromosome 9q34.3 that eliminate one \u003cem\u003eEHMT1\u003c/em\u003e copy, most common forms of \u003cem\u003eEHMT1\u003c/em\u003e loss-of-function reported in KS patients are single nucleotide changes that cause frameshifts. Missense mutations are rarer and only a couple have been properly validated\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Larger efforts would be needed to evaluate the effects of \u003cem\u003eEHMT1\u003c/em\u003e and \u003cem\u003eEHMT2\u003c/em\u003e missense variants and their consequences for complex function and disease severity.\u003c/p\u003e \u003cp\u003eRegarding diagnostic perspectives for patients with \u003cem\u003eEHMT2\u003c/em\u003e alterations, we found a DNA methylation signature for patient ND095 that resembles KS patients, likely due to the formation of a complex between EHMT1 and EHMT2. This shared, but slightly different, DNA methylation profile has been observed also for other disorders of the same protein complex such as BAFopathies\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, the increased number of patients with rare diseases tested for DNA methylation profiles has improved the resolution and specificity of the episignatures ranging from altered protein complexes to genes, protein domains, and even single nucleotides\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Therefore, it is possible that a specific DNA methylation episignature for EHMT2 loss-of-function, different from that of patients with KS, can be established in the future after the identification and profiling of more patients with pathogenic \u003cem\u003eEHMT2\u003c/em\u003e alterations.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study used tools provided through the RD-Connect GPAP, which received funding originally from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 305444. We thank the Bioinformatics Unit at ISCIII (I. Cuesta and S. Monzon) for their help in exome analysis, and A. Krepischi and L. Machado for discussions. We also thank the patients\u0026rsquo; association Kleefstra Espa\u0026ntilde;a, the National Biobank of Rare Diseases (BioNER), the patients, their families, and their physicians, as well as the entire SpainUDP consortium. The authors appreciate the support of the Undiagnosed Diseases Network International (UDNI) for data sharing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded with project PID2021-128087OB-I00 by MCIN /AEI /10.13039/501100011033 / FEDER, UE to M.J.B, and AESI PT20CIII/00009 (ISCIII Platform of Biobanks and Biomodels PT-20). Funding was also partially provided by the Genome Canada and the Ontario Genomics Institute Genomics Applications Partnership Program Grant (OGI-188).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMD and MJB conceived and designed the work. BMD, ELM, JG, EBS, MP, PMR, RCC, DR, TK, JK, JR, BS and MJB participated in data collection. JK, JR, BS and MJB participate in data analysis. PMR, RCC, LLJ, LMM, MFP, EHS, GGM, BB and DSP generated critical reagents for the study. \u0026nbsp;ABN, MHM and MJB carried out functional assays.\u0026nbsp;MJB coordinated the research and wrote the manuscript with input from all authors. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consents were signed by patient\u0026rsquo;s legal representatives. This research project was approved by the ISCIII Research Ethics Committee entry number CEI PI 03_2022.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interest\u003c/p\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eRNA-seq raw data is available upon request to the corresponding author. The pathogenic EHMT2 NM_006709.5:c.3229G \u0026gt; T variant detected in our study has been submitted to ClinVar (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/clinvar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, submission number: SUB14159160).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKleefstra T, Brunner HG, Amiel J \u003cem\u003eet al.\u003c/em\u003e Loss-of-Function Mutations in Euchromatin Histone Methyl Transferase 1 (EHMT1) Cause the 9q34 Subtelomeric Deletion Syndrome. 2006www.ajhg.org.\u003c/li\u003e\n\u003cli\u003eWillemsen MH, Vulto-Van Silfhout AT, Nillesen WM \u003cem\u003eet al.\u003c/em\u003e Update on Kleefstra syndrome. \u003cem\u003eMol Syndromol\u003c/em\u003e 2012; \u003cstrong\u003e2\u003c/strong\u003e: 202\u0026ndash;212.\u003c/li\u003e\n\u003cli\u003eKleefstra T, Kramer JM, Neveling K \u003cem\u003eet al.\u003c/em\u003e Disruption of an EHMT1-associated chromatin-modification module causes intellectual disability. \u003cem\u003eAm J Hum Genet\u003c/em\u003e 2012; \u003cstrong\u003e91\u003c/strong\u003e: 73\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eFaundes V, Newman WG, Bernardini L \u003cem\u003eet al.\u003c/em\u003e Histone Lysine Methylases and Demethylases in the Landscape of Human Developmental Disorders. \u003cem\u003eAm J Hum Genet\u003c/em\u003e 2018; \u003cstrong\u003e102\u003c/strong\u003e: 175\u0026ndash;187.\u003c/li\u003e\n\u003cli\u003eKoemans TS, Kleefstra T, Chubak MC \u003cem\u003eet al.\u003c/em\u003e Functional convergence of histone methyltransferases EHMT1 and KMT2C involved in intellectual disability and autism spectrum disorder. \u003cem\u003ePLoS Genet\u003c/em\u003e 2017; \u003cstrong\u003e13\u003c/strong\u003e: e1006864.\u003c/li\u003e\n\u003cli\u003eTachibana M, Sugimoto K, Fukushima T, Shinkai Y. 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One such syndrome is Kleefstra syndrome (KS), which results from heterozygous alterations in the \u003cem\u003eEHMT1\u003c/em\u003e gene, leading to loss of function. EHMT1 and EHMT2 are highly similar histone methyltransferases that play crucial roles in development. Despite their similarity, alterations in \u003cem\u003eEHMT2\u003c/em\u003e have not been previously reported. In this study, we present a pediatric patient exhibiting a phenotype overlapping with KS, harboring a de novo single base substitution in EHMT2. This substitution results in the amino acid change p.Ala1077Ser in the catalytic SET domain, causing a decrease in the affinity of this domain for histone H3 tail and a three- to five-fold reduction in enzyme activity. As part of an advanced diagnostic strategy, we leveraged epigenomics and proteomics data to comprehensively characterize the EHMT2 p.Ala1077Ser variant. Analysis of DNA methylation, histone methylation, and gene expression profiles reveals a substantial overlap between the EHMT2 p.Ala1077Ser variant and KS. Based on these findings, we propose that \u003cem\u003eEHMT2\u003c/em\u003e haploinsufficiency leads to a Kleefstra-like syndrome. While we cannot entirely rule out dominant negative effects caused by the EHMT2 p.Ala1077Ser variant, our data, in conjunction with previously published studies, suggest that the loss of EHMT2 function is more detrimental to cells than the loss of EHMT1. This may explain the rarity of individuals with alterations in \u003cem\u003eEHMT2\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Ehmt2 Loss-of-function Alterations Cause a Kleefstra-like Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-14 18:34:13","doi":"10.21203/rs.3.rs-3893528/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e11bb900-d78b-404b-b02c-4dbdb5fe74a3","owner":[],"postedDate":"February 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-17T14:28:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-14 18:34:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3893528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3893528","identity":"rs-3893528","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}