HGNC Approved Gene Symbol: DNMT3A
SNOMEDCT: 768843007;
Cytogenetic location: 2p23.3 Genomic coordinates (GRCh38) : 2:25,227,874-25,342,590 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 2p23.3 | Acute myeloid leukemia, somatic | 601626 | 3 | |
| Heyn-Sproul-Jackson syndrome | 618724 | Autosomal dominant | 3 | |
| Tatton-Brown-Rahman syndrome | 615879 | Autosomal dominant | 3 |
Mammalian cells can epigenetically modify their genomes via DNA methylation. DNA methylation plays important roles in genomic imprinting and X-chromosome inactivation and is essential for mammalian development. DNMT3A appears to function as a de novo methyltransferase because it can methylate unmethylated and hemimethylated DNA with equal efficiencies (Yanagisawa et al., 2002).
Aberrant de novo methylation of growth regulatory genes is associated with tumorigenesis in humans (Baylin et al., 1998). However, Lei et al. (1996) showed that de novo methylation persists in embryonic stem (ES) cells lacking Dnmt1 (126375), which encodes the constitutive DNA methyltransferase-1, indicating the existence of independently encoded de novo methyltransferases. By a searching an EST database using full-length bacterial type II cytosine-5 methyltransferase sequences as queries, followed by isolation and sequencing of overlapping cDNA clones, Okano et al. (1998) identified 2 homologous genes in both human and mouse that contain the highly conserved cytosine-5 methyltransferase motifs. The mouse genes, termed Dnmt3a and Dnmt3b (602900), show little sequence similarity to mouse Dnmt1 and Dnmt2 (602478), and masc1 from Ascobolus. The Dnmt3a cDNA encodes a protein of 908 amino acids. The human DNMT3A and DNMT3B cDNAs are highly homologous to the mouse genes. Dnmt3a and Dnmt3b transcripts were abundantly expressed in undifferentiated embryonic stem cells.
Using Northern blot analysis, Xie et al. (1999) detected major DNMT3A transcripts of about 4.0, 4.4, and 9.5 kb in all tissues examined except small intestine.
Robertson et al. (1999) detected DNMT3A expression in all adult and fetal tissues examined by Northern blot analysis. Expression was highest in fetal tissues and was extremely high in fetal liver. Semiquantitative RT-PCR confirmed ubiquitous DNMT3A expression. Expression of DNMT1, DNMT3A, and DNMT3B appeared to be coregulated in most tissues, since they frequently had a similar pattern of expression.
Weisenberger et al. (2002) identified splice variants of mouse and human DNMT3A containing an alternate noncoding upstream exon 1 that they called exon 1-beta. Transcripts containing either exon 1-alpha or exon 1-beta were predicted to encode the same protein. Weisenberger et al. (2002) also identified variants containing portions of intron 4, and these transcripts were predicted to encode a truncated protein. In all human cell lines and tissues examined, RT-PCR showed higher expression of transcripts containing exon 1-alpha or lacking intron 4 compared with those containing exon 1-beta or intron 4.
Chen et al. (2002) identified a small isoform of DNMT3A, designated DNMT3A2, in human and mouse tissues. The DNMT3A2 transcript is initiated within the sixth intron of the DNMT3A gene and encodes a protein that lacks the N-terminal 223 (in human) or 219 (in mouse) amino acid residues of the full-length protein. Although recombinant DNMT3A2 showed cytosine methyltransferase activity, it showed different subcellular localization compared to DNMT3A: DNMT3A was concentrated on heterochromatin, whereas DNMT3A2 showed a pattern suggesting localization to transcriptionally active euchromatin. DNMT3A2 was the predominant isoform in embryonic stem cells and embryonal carcinoma cells, and was also detected in testis, ovary, thymus, and spleen, whereas DNMT3A was expressed ubiquitously at low levels. The findings suggested that the 2 isoforms may have distinct DNA targets and different functions in development.
Using reverse RNA dot blot analysis of human fetal gonads, Galetzka et al. (2007) found that expression of DNMT1 and DNMT3A peaked in mitotically quiescent human fetal spermatogonia around 21 weeks' gestation. In fetal ovary, upregulation of DNMT1 and DNMT3A mRNA occurred during a very brief period at 16 weeks' gestation, when oocytes proceeded through meiotic prophase. The most abundant DNMT3A variant in fetal testis and ovary was DNMT3A2. In both male and female fetal gonads, expression of MBD2 (603547) and MBD4 (603574) was tightly linked to DNMT expression, suggesting that concomitant upregulation of DNMT1, DNMT3A, MBD2, and MBD4 is associated with prenatal remethylation in the male and female germ line.
Gu et al. (2022) noted that there are 2 main protein isoforms of mouse Dnmt3a resulting from alternative splicing. The longer isoform, Dnmt3a1, contains 908 amino acids and has an N-terminal domain, a PWWP domain, an ADD domain, and a C-terminal catalytic methyltransferase domain. The shorter isoform, Dnmt3a2, contains 689 amino acids and lacks the 219-amino acid N terminus of Dnmt3a1. The isoforms are differentially expressed from stem cells to somatic tissues. Analyses with RT-PCR and a knockin C-terminally Flag-tagged Dnmt3a showed that Dnmt3a1 was more highly expressed in most mouse tissues, particularly in brain regions, and that Dnmt3a2 was expressed moderately in thymus and weakly in bone marrow, spleen, and testis.
Weisenberger et al. (2002) determined that the DNMT3A gene contains 26 exons, including alternative first exons (exons 1-alpha and 1-beta) and exons 7a and 7b. Exons 1-alpha and 1-beta are both located in a CpG-rich region. The upstream exon 1, exon 1-beta, may contain a short ORF that ends at a stop codon in exon 2. DNMT3A also has alternate polyadenylation sites.
Yanagisawa et al. (2002) identified 3 untranslated alternative first exons (exons 1A, 1B, and 1C) in the DNMT3A gene, each of which is associated with a unique promoter region. There are also 4 transcriptional start sites, 2 of which are in exon 1B. The translational start codon is located within exon 2. All promoter regions lack TATA sequences, and the promoters associated with exons 1A and 1B are CpG rich, while that associated with exon 1C is relatively CpG poor.
By FISH, Xie et al. (1999) mapped the DNMT3A gene to 2p23. Robertson et al. (1999) also mapped the DNMT3A gene to 2p23 using FISH.
Crystal Structure
Jia et al. (2007) used crystallography to show that the C-terminal domain of human DNMT3L (606588) interacts with the catalytic domain of DNMT3A, demonstrating that DNMT3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complex C-terminal domains of DNMT3A and DNMT3L showed further dimerization through DNMT3A-DNMT3A interaction, forming a tetrameric complex with 2 active sites. Substitution of key noncatalytic residues at the DNMT3A-DNMT3L interface or the DNMT3A-DNMT3A interface eliminated enzymatic activity. Molecular modeling of a DNA-DNMT3A dimer indicated that the 2 active sites are separated by about 1 DNA helical turn. The C-terminal domain of DNMT3A oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of DNMT3A on long DNA revealed a correlation of methylated CpG sites at distances of 8 to 10 basepairs, indicating that oligomerization leads DNMT3A to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. Jia et al. (2007) concluded that their results suggested a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.
Guo et al. (2015) determined the crystal structures of DNMT3A/DNMT3L (autoinhibitory form) and DNMT3A/DNMT3L-H3 (active form) complexes at 3.82- and 2.90-angstrom resolution, respectively. Structural and biochemical analyses indicated that the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A interacts with and inhibits enzymatic activity of the catalytic domain through blocking its DNA-binding affinity. Histone H3 (see 602810) (but not H3K4me3) disrupts ADD-catalytic domain interaction, induces a large movement of the ADD domain, and thus releases the autoinhibition of DNMT3A. The authors concluded that the finding adds another layer of regulation of DNA methylation to ensure that the enzyme is mainly activated at proper targeting loci when unmethylated H3K4 is present, and strongly supports a negative correlation between H3K4me3 and DNA methylation across the mammalian genome.
Zhang et al. (2018) reported the 2.65-angstrom crystal structure of the DNMT3A-DNMT3L-DNA complex in which 2 DNMT3A monomers simultaneously attack 2 cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 basepairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Hematologic cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Zhang et al. (2018) concluded that their study revealed the mechanistic basis for DNMT3A-mediated DNA methylation and established its etiologic link to human disease.
Using a deep enzymology approach combined with cellular methylome profiling, Gao et al. (2020) characterized the flanking sequence preferences of DNMT3A and DNMT3B, which manifested more than 100-fold different methylation rates of CpG sites across different sequence contexts. Subsequently, the authors determined the crystal structures of DNMT3B in complex with both CpG and CpA DNA. A hydrogen bond in the catalytic loop of DNMT3B caused a lower CpG specificity than DNMT3A, whereas the interplay of target recognition domain and homodimeric interface fine-tuned the distinct target selection between the 2 enzymes, with lys777 of DNMT3B acting as a unique sensor of the +1 flanking base. The findings revealed distinctive substrate-readout mechanisms of the 2 enzymes.
Okano et al. (1998) performed experiments suggesting that mouse Dnmt3a and Dnmt3b encode the long-sought de novo DNA methyltransferases.
Using coimmunoprecipitation of recombinant proteins expressed in insect cells and COS-7 cells, Kim et al. (2002) identified interaction between DNMT1, DNMT3A, and DNMT3B. DNMT3A and DNMT3B were also able to form complexes in the absence of DNMT1. By mutation analysis, they localized the interacting domains to the N termini of the proteins. Immunocytochemical staining revealed mostly nuclear colocalization of fluorescence-labeled proteins, except for DNMT3A, which was found either exclusively in the cytoplasm or in both the cytoplasm and nucleus. In vivo coexpression of DNMT1 and DNMT3A and/or DNMT3B led to methylation spreading in the genome, suggesting cooperation between them.
Vire et al. (2006) showed that the silencing pathways of the polycomb group (PcG) and DNA methyltransferases systems are mechanically linked. They found that the PcG protein EZH2 (601573) interacts--within the context of the polycomb repressive complexes 2 and 3 (PRC2/3)--with DNA methyltransferases DNMT1 (126375), DNMT3A, and DNMT3B and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicated that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, Vire et al. (2006) showed by bisulfite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Vire et al. (2006) concluded that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between 2 key epigenetic repression systems.
Using mass spectrometry, Ooi et al. (2007) identified the main proteins that interacted in vivo with the product of an epitope-tagged allele of the endogenous DNMT3L (606588) gene as DNMT3A2, DNMT3B (602900), and the 4 core histones. Various studies indicated that DNMT3L recognizes histone H3 tails that are unmethylated at lysine-4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.
The microRNAs MIRN29A (610782), MIRN29B (see MIRN29B1; 610783), and MIRN29C (610784) are downregulated in lung cancer. Fabbri et al. (2007) identified complementarity sites for the MIRN29s in the 3-prime UTRs of DNMT3A and DNMT3B, which are frequently upregulated in lung cancers with poor prognosis. Expression of the MIRN29s was inversely correlated with levels of DNMT3A and DNMT3B in lung cancer tissues, and the MIRN29s directly targeted DNMT3A and DNMT3B. Enforced expression of MIRN29s in lung cancer cell lines restored normal patterns of DNA methylation, induced reexpression of methylation-silenced tumor suppressor genes, and inhibited tumorigenicity in vitro and in vivo.
Using ELISA, Balada et al. (2008) determined that the DNA deoxymethylcytosine content of purified CD4 (186940)-positive T cells was lower in patients with systemic lupus erythematosus (SLE; 152700) than in controls. RT-PCR analysis detected no differences in DNMT1, DNMT3A, or DNMT3B transcript levels between SLE patients and controls. However, simultaneous association of low complement counts with lymphopenia, high titers of anti-double-stranded DNA, or a high SLE disease activity index resulted in an increase in at least 1 of the DNMTs. Balada et al. (2008) proposed that patients with active SLE and DNA hypomethylation have increased DNMT mRNA levels.
Wu et al. (2010) showed that the de novo DNA methyltransferase Dnmt3a is expressed in mouse postnatal neural stem cells and is required for neurogenesis. Genomewide analysis of postnatal neural stem cells indicated that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent nonproximal promoter methylation promoted expression of these neurogenic genes by functionally antagonizing polycomb (see 603079) repression. Thus, Wu et al. (2010) concluded that nonpromoter DNA methylation by Dnmt3a may be used for maintaining active chromatin states of genes critical for development.
Smallwood et al. (2011) identified over a thousand methylated CpG islands in mature mouse oocytes. Both Dnmt3a -/- and Dnmt3l -/- oocytes showed a gross, genomewide reduction in CpG methylation, including at repetitive elements and CpG islands independent of their genic location. Smallwood et al. (2011) concluded that DNMT3A and DNMT3L have a genomewide role in CpG island methylation beyond genomic imprinting.
Chen et al. (2012) found that mouse de novo DNA methyltransferases Dnmt3a and Dnmt3b also possess redox-dependent DNA dehydroxymethylases to convert 5-hydroxymethyl cytosine (5-hmC) to C. Examination of the in vitro DNA dehydroxymethylation activity in nuclear extracts from 293T cells exogenously expressing Dnmt3a and Dnmt3b as well as their mutant forms with amino acid substitutions at the catalytic site of C methylation showed the requirement of intact DNA 5-mC catalytic sites of both enzymes for their dehydroxymethylase activities. Characterization of the methylation and dehydroxymethylation activities of recombinant human DNMT3A and mouse Dnmt3b demonstrated that both can function as dose-dependent DNA dehydroxymethylases. Although the dehydroxymethylation and methylation reactions likely utilize the same catalytic site, the redox state of the enzymes plays a crucial role in determining function as methyltransferases or dehydroxymethylases.
Dai et al. (2016) demonstrated that inactivation of all 3 Tet genes (see TET1, 607790) in mice leads to gastrulation phenotypes, including primitive streak patterning defects in association with impaired maturation of axial mesoderm and failed specification of paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal (601265) signaling. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signaling contributes to the gastrulation failure of Tet mutants. Increased Nodal signaling is probably due to diminished expression of the Lefty1 (603037) and Lefty2 (601877) genes, which encode inhibitors of Nodal signaling. Moreover, reduction in Lefty gene expression is linked to elevated DNA methylation, as both Lefty-Nodal signaling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b (602900) genes are disrupted. Additionally, a point mutation in Tet that specifically abolishes the dioxygenase activity causes similar morphologic and molecular abnormalities as the null mutation. Dai et al. (2016) concluded that TET-mediated oxidation of 5-methylcytosine modulates Lefty-Nodal signaling by promoting demethylation in opposition to methylation by DNMT3A and DNMT3B. The authors also concluded that their findings revealed a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation crucial to regulation of key signaling pathways in early body plan formation.
Weinberg et al. (2019) reported that NSD1 (606681)-mediated H3K36me2 is required for the recruitment of DNMT3A and maintenance DNA methylation at intergenic regions. Genomewide analysis showed that the binding and activity of DNMT3A colocalize with H3K36me2 at noncoding regions of euchromatin. Genetic ablation of Nsd1 and its paralog Nsd2 in mouse cells resulted in a redistribution of Dnmt3A to H3K36me3-modified gene bodies and a reduction in the methylation of intergenic DNA. Blood samples from patients with Sotos syndrome (117550) and NSD1-mutant tumors also exhibited hypomethylation of intergenic DNA. The PWWP domain of DNMT3A showed dual recognition of H3K36me2 and H3K36me3 in vitro, with a higher binding affinity towards H3K36me2 that was abrogated by Tatton-Brown-Rahman syndrome (TBRS; 615879)-derived missense mutations. Weinberg et al. (2019) concluded that their study revealed a trans-chromatin regulatory pathway that connects aberrant intergenic CpG methylation to human neoplastic and developmental overgrowth.
Using purified recombinant proteins, Sandoval and Reich (2019) showed that wildtype p53 (TP53; 191170) inhibited DNA methylation activity of human DNMT3A. The inhibitory effect of p53 on DNA methylation was specific to human DNMT3A, as p53 did not inhibit the DNA methylation activity of the bacterial homolog of DNMT3A. p53 decreased DNMT3A activity by forming a heterotetramer with DNMT3A, and mutation mapping suggested that p53 interacted with the tetramer interface of DNMT3A, with R736 on the DNMT3A tetramer interface contributing to the necessary contacts for p53 inhibition of DNMT3A. The DNMT3A tetramer interface also interacted with DNMT3L, but DNMT3A had a higher affinity for p53, and regulation of DNMT3A by p53 was dominant over that of DNMT3L. p53 interacted with DNMT3A to form heterotetramers, and inhibition of DNMT3A DNA methylation activity by p53 did not arise through disruption of DNMT3A binding to DNA. Additionally, 2 hotspot mutations in the DNA-binding region of p53 showed levels of DNMT3A inhibition comparable to that of wildtype p53, but they showed altered regulation of DNMT3A in the presence of DNMT3L, supporting the notion that the regulatory effect of DNMT3L on DNMT3A activity was dominant over that of these p53 mutants.
Using knockout analysis in mouse embryonic fibroblasts, Yagi et al. (2020) showed that Dnmt3a was exclusively required for de novo methylation at both transcription start site regions and gene bodies of PcG target developmental genes, whereas Dnmt3b had a predominant role on the X chromosome during X chromosome inactivation. Gene ontology analysis revealed that Dnmt3b-specific genes were involved in synaptonemal complex assembly, plasma membrane function, and cell proliferation in forebrain. By contrast, Dnmt3a-specific genes were involved in sequence-specific DNA binding, transcription, and pharyngeal system development. Dnmt3l was dispensable for Dnmt3a-mediated methylation at PcG target genes during embryonic development. Further analysis indicated that Dnmt3a-mediated DNA methylation played a role in stable silencing of a subset of PcG target developmental genes in a tissue-specific manner during development. Patients with DNMT3 mutations exhibited reduced DNA metylation at regions that were hypomethylated in mouse Dnmt3-knockout cells, suggesting that the region specificity of de novo methylation by DNMT3A and DNMT3B is essentially shared between mice and humans.
The localization of DNMT3A is facilitated by its PWWP domain, which recognizes histone H3 lysine-36 (H3K36) methylation; normally it is not localized at CpG islands. However, Weinberg et al. (2021) found that DNMT3A with PWWP domain mutants K299I, R318W, W330R, and D333N, identified in patients with paragangliomas and microcephalic dwarfism, lost its PWWP domain H3K36 reader functionality and localized to CpG islands. Localization of DNMT3A mutants to CGIs required the presence of PRC1 (603484)-catalyzed monoubiquitylated histone H2A lysine-119 (H2AK119ub) but was irrespective of the amounts of H3K27me3. DNMT3A interacted directly with H2AK119ub-modified nucleosomes through a putative N-terminal ubiquitin-dependent recruitment region (UDR), which allowed the DNMT3A PWWP domain mutants to be recruited to H2AK119ub-enriched CpG islands and led to DNA hypermethylation at CpG islands. Thus, for wildtype DNMT3A, recruitment by the PWWP domain competed with DNMT3A recruitment by the UDR to maintain the dynamics of DNA methylation at PRC1-targeted CpG islands.
Tatton-Brown-Rahman Syndrome
In 13 unrelated patients with Tatton-Brown-Rahman syndrome (TBRS; 615879), characterized by tall stature, a distinctive facial appearance, and intellectual disability, Tatton-Brown et al. (2014) identified 13 different de novo heterozygous mutations in the DNMT3A gene (see, e.g., 602769.0001-602769.0005). The first 2 mutations were found by exome sequencing of 10 patients with an overgrowth disorder, and the subsequent 11 mutations were found by sequencing the DNMT3A gene in 152 patients with an overgrowth disorder. The mutations altered residues in functional domains of the protein, and protein modeling suggested that they may interfere with domain-domain interactions and histone binding, thereby disrupting de novo methylation. However, in vitro functional studies were not performed.
In a 6-year-old girl with TBRS, Kosaki et al. (2017) identified a heterozygous germline missense mutation (R882H; 602769.0006) in the DNMT3A gene. The authors noted that the arg882 residue is a somatic mutation hotspot for acute myelogeneous leukemia (AML; see 601626).
In 3 unrelated patients with TBRS, Shen et al. (2017) identified 2 different heterozygous mutations at the arg882 residue in the DNMT3A gene: R882H and R882C (602769.0007). Shen et al. (2017) stated that these variants appear in exome databases at frequencies suggesting that they are not pathogenic; however, further investigation revealed that the median variant allele fraction in individuals reported with these variants was 19%, strongly suggesting that the observed frequencies are due to confounding of somatic variants in individuals with age-related clonal hematopoiesis.
In a 6-year-old girl (patient 5), who was previously reported by DeMari et al. (2016) at age 3.5 years with global developmental delay (MRD56; 617854) and a heterozygous mutation in the CLTC gene (118955.0001), Balci et al. (2020) identified heterozygosity for the recurrent R882C mutation in the DNMT3A gene. At age 6 years, the child presented with lymphadenopathy, a mediastinal mass, and hypercalcemia, and was diagnosed with T-cell lymphoblastic lymphoma.
Heyn-Sproul-Jackson syndrome
In 3 unrelated patients with Heyn-Sproul-Jackson syndrome (HESJAS; 618724), Heyn et al. (2019) identified de novo heterozygous missense mutations in the conserved PWWP domain of the DNMT3A gene (W330R, 602769.0008 and D333N, 602769.0009). The mutations, which were found by whole-exome sequencing and confirmed by Sanger sequencing, were not found in large databases such as gnomAD or ExAC. In vitro functional expression studies using recombinant proteins showed that the W330R mutant was unable to bind H3K36me2 or H3K36me3, whereas wildtype DNMT3A was able to bind these histone peptides. The D333N mutation was predicted to have a similar effect. Analysis of patient-derived fibroblasts and peripheral blood leukocytes showed that the variants had altered chromatin binding specificity resulting in significant DNA hypermethylation of multiple genes compared to controls. Gene ontology analysis indicated that most of the hypermethylated regions likely affected genes associated with transcription and developmental processes, including HOX genes. Chromatin immunoprecipitation studies of normal cells showed that the affected differentially methylated regions (DMRs) contained CpG islands and were associated with polycomb repressive complexes (PRCs). However, regions of increased methylation in patient cells were not confined to CpG islands and extended throughout normally hypomethylated regions, termed 'DNA methylation valleys.' Patient fibroblasts showed reduced H3K27me3 levels at the DMRs compared to controls, suggesting disruption of PRC2 binding and activity. Expression of the orthologous W326R mutation in murine embryonic stem cells caused DNA hypermethylation at the DMRs during differentiation to embryoid bodies and to neural progenitor cells; RNA sequencing of these mutant murine cells and W330R fibroblasts showed abnormal expression of transcription factors that tended to skew toward differentiation and away from self-renewal. Heterozygous mutant mice had reduced brain size and body weight, and were proportionately small compared to controls. Hypermethylation at the DMRs were observed in neurons in the mouse cerebral cortex. Heyn et al. (2019) concluded that the DNMT3A mutations result in a gain-of-function effect, causing hypermethylation at polycomb-marked developmental genes during early stages of cell fate specification and differentiation. The authors noted that mutations affecting other regions of this gene result in a macrocephalic overgrowth syndrome (TBRS; 615879), which is the opposite of the phenotype observed in their patients.
Somatic Mutations in Acute Myeloid Leukemia
Ley et al. (2010) found that leukemia samples from 62 (22.1%) of 281 patients with acute myelogenous leukemia (AML; 601626) had somatic mutations in the DNMT3A gene that were predicted to affect translation. The most common missense mutations, found in 37 patients, affected amino acid arg882 (see, e.g., 602769.0006 and 602769.0007). DNMT3A mutations were enriched in 33.7% of patients with an intermediate-risk cytogenetic profile, but were absent in all 79 patients with a favorable-risk cytogenetic profile. However, analysis of AML tumor samples showed no difference in DNMT3A expression between those with mutations and those without mutations. In addition, there was no difference in 5-methylcytosine content, global patterns of methylation, or other gene expression between AML genomes carrying DNMT3A mutations and those without mutations, with a small number of exceptions: some regions had significantly reduced methylation, but no consistent correlations could be observed. The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months compared to 41.1 months). DNMT3A mutations were associated with adverse clinical outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 (136351) mutations. The findings suggested that DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome.
Yan et al. (2011) reported the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). Yan et al. (2011) discovered mutations in DNMT3A in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 (see 602810) in vitro. Notably, there were alterations in DNA methylation patterns and/or gene expression profiles (such as HOXB genes, e.g., 142960) in samples with DNMT3A mutations as compared to those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases.
Walter et al. (2011) identified 13 somatic heterozygous mutations in the DNMT3A gene in 8% of bone marrow samples derived from 150 patients with myelodysplastic syndrome (MDS; 614286). Four of the mutations occurred at residue arg882, in the methyltransferase domain. Only 2 of the mutations resulted in truncation, and mRNA expression of the missense mutations was similar to wildtype. Although the survival of patients with DNMT3A mutations was worse than of those without these mutations, the overall sample was small. In all, 58% of patients with a DNMT3A mutation progressed to AML, compared to 28% without a mutation. Analysis of the bone marrow cells showed that the mutations were present in nearly all of the cells, although the myeloblast count was less than 30% for most samples, suggesting that DNMT3A mutations are very early genetic events in MDS and may confer a clonal advantage to cells with the mutation. The findings also indicated that epigenetic changes contribute to MDS pathogenesis.
The Cancer Genome Atlas Research Network (2013) analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA methylation analysis. The authors identified recurrent mutations in the DNMT3 gene in 51/200 (26%) samples.
Brewin et al. (2013) noted that the study of the Cancer Genome Atlas Research Network (2013) did not reveal which mutations occurred in the founding clone, as would be expected for an initiator of disease, and which occurred in minor clones, which subsequently drive disease. Miller et al. (2013) responded that genes mutated almost exclusively in founding clones in their study included DNMT3A (38 of 40 mutations in founding clones). They identified several other genes that contained mutations they considered probable initiators, and other genes mutations in which were considered probably cooperating mutations.
Shlush et al. (2014) found recurrent DNMT3A mutations at high allele frequency in highly purified hematopoietic stem cells (HSCs) as well as progenitor and mature cell fractions from the blood of AML (601626) patients, but these cells did not have the coincident NPM1 (164040) mutations present in AML blasts. DNMT3A mutation-bearing HSCs showed a multilineage repopulation advantage over nonmutated HSCs in xenografts, establishing their identity as preleukemic HSCs. Preleukemic HSCs were found in remission samples, indicating that they survive chemotherapy. Shlush et al. (2014) concluded that DNMT3A mutations arise early in AML evolution, probably in HSCs, leading to a clonally expanded pool of preleukemic HSCs from which AML evolves.
Associations Pending Confirmation
In a 22-year-old woman with multiple laryngeal paragangliomas and a right carotid paraganglioma, and jugulotympanic paragangliomas that developed later, Remacha et al. (2018) identified a de novo germline missense mutation in DNMT3A (c.896A-T, lys299 to ile, K299I) affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. The patient had no family history of pheochromocytoma or paraganglioma. Remacha et al. (2018) then sequenced exons 8 and 9 of DNMT3A in 35 patients without germline alterations in known susceptibility genes, but who were strongly suspected of having hereditary pheochromocytoma/paraganglioma due to presence of multiple tumors and/or family history. One additional missense variant, arg318 to trp (R318W, c.952C-T), was found in germline DNA from a 54-year-old woman with 2 head and neck paragangliomas and a family history of disease. Subsequent analysis of 94 paragangliomas identified the K299I variant in several tumors. Knockin of the K299I mutation in HeLa cells showed that the mutation increased DNMT3A DNA methylation capabilities. Additional functional studies showed that both DNMT3A mutations led to similar characteristic methylation profiles independent of the tissue of origin.
Kim et al. (2021) analyzed data from the UK Biobank database and found that clonal hematopoiesis of indeterminate potential (CHIP) was associated with an increased incidence of osteoporosis. Further analysis demonstrated that the most commonly mutated gene in CHIP was in DNMT3A, and mutations were associated with lower estimated bone marrow density on heel ultrasound.
The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. Lyko et al. (1999) expressed Dnmt1 and Dnmt3a in transgenic Drosophila melanogaster. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When coexpressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development. The expression of Dnmt3a but not Dnmt1 caused developmental defects in Drosophila, with the majority dying in the pupal stage. Tissue-specific expression of Dnmt3a in the Drosophila eye resulted in small or absent eyes.
Okano et al. (1999) generated mice with targeted disruption of the Dnmt3a and Dnmt3b genes. Inactivation of both genes blocked de novo methylation in embryonic stem cells and early embryos but had no effect on maintenance of imprinted methylation patterns. Dnmt3a -/- mice developed to term and appeared to be normal at birth. However, most homozygous mutant mice became runted and died at about 4 weeks of age. In contrast, no viable Dnmt3b -/- mice were recovered at birth. Dissection of embryos at different stages of development revealed that Dnmt3b -/- embryos had multiple developmental defects, including growth impairment and rostral neural tube defects with variable severity at later stages of development, though most of them appeared to develop normally before E9.5. Dnmt3a and Dnmt3b also exhibited nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. These results indicated that both Dnmt3a and Dnmt3b are required for genomewide de novo methylation and are essential for mammalian development.
Kaneda et al. (2004) reported the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females died in utero and lacked methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males showed impaired spermatogenesis and lacked methylation at 2 of 3 paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring showed no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at 1 locus (IG-DMR). Kaneda et al. (2004) concluded that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but other factors are probably involved.
Miller and Sweatt (2007) found that DNA methylation mediated by DNMTs was dynamically regulated during learning and memory consolidation in adult rats. Animals exposed to an associative context plus shock showed increased Dnmt3a and Dnmt3b mRNA in hippocampal area CA1 compared to context-only animals. Context plus shock rats showed increased methylation and decreased mRNA of the memory suppressor gene PP1C-beta (PPP1CB; 600590) compared to shock-only controls, as well as increased demethylation and increased mRNA levels of reelin (RELN; 600514), a gene involved in synaptic plasticity, compared to controls. The methylation levels of both these target genes returned to baseline within a day, indicating rapid and dynamic changes. Treatment with a DNMT inhibitor blocked the methylation changes and prevented memory consolidation of fear-conditioned learning, but the memory changes were plastic, and memory consolidation was reestablished after the inhibitor wore off. Miller and Sweatt (2007) noted that DNA methylation has been viewed as having an exclusive role in development, but they emphasized that their findings indicated that rapid and dynamic alteration of DNA methylation can occur in the adult central nervous system in response to environmental stimuli during associative learning in the hippocampus.
In mice, Challen et al. (2012) found that Dnmt3a was highly expressed in the most primitive long-term hematopoietic stem cells. Conditional knockout of the murine Dnmt3a gene in somatic hematopoietic stem cells resulted in a significant increase (about 7-fold) in hematopoietic stem cells in the bone marrow after 18 weeks. Although these changes were accompanied by an increase in peripheral blood production, blood production was not proportional to the increase of stem cells in the bone marrow, suggesting a defect in differentiation. The loss of Dnmt3a progressively impaired stem cell differentiation over serial transplantation, while simultaneously expanding hematopoietic stem cells in the bone marrow, favoring self-renewal rather than differentiation. Methylation analysis showed that loss of Dnmt3A caused both hypo- and hypermethylation at distinct loci compared to controls; however, most CpG-rich islands were hypermethylated. Challen et al. (2012) suggested that the paradoxical finding of increased methylation in these cells may reflect indirect changes. Global transcriptional profiling showed upregulation of hematopoietic stem cell multipotency genes and downregulation of differentiation genes. The cells that did differentiate despite loss of Dnmt3a showed a reduction in global DNA methylation, as well as hypomethylated sites that correlated with increased expression of genes normally restricted to hematopoietic stem cells. None of the Dnmt3a-mutant mice developed leukemia, suggesting that other factors are involved in oncogenic changes.
In mice, Oliveira et al. (2012) found that Dnmt3a2, but not Dnmt3a1, was activated by neuronal activity in both an NMDA receptor- and calcium signaling-dependent manner. Learning induced the expression of Dnmt3a2 in the hippocampus of young mice, but learning-induced activation of Dnmt3a2 was impaired in the hippocampus of aged mice. In addition, the levels of Dnmt3a1 and Dnmt3a2 transcripts were reduced in the hippocampus and cortex of aged mice compared to young mice. Viral-mediated overexpression of Dnmt3a2 in the hippocampus of aged mice resulted in a significant increase in global DNA methylation levels, and these mice showed improved cognitive memory abilities compared to control aged mice in several different tests. Suppression of the Dnmt3a2 isoform in young mice resulted in defects in long-term memory, but did not appear to affect short-term memory. The results suggested a role for Dnmt3a2-mediated DNA methylation in changes in cognitive abilities associated with aging.
In Npm1C (164040)/Dnmt3a mutant knockin mice, a model of AML development, leukemia is preceded by a period of extended myeloid progenitor cell proliferation and self-renewal. Uckelmann et al. (2020) found that this self-renewal can be reversed by oral administration of a small molecule that targets the MLL1-Menin (159555/613733) chromatin complex. Uckelmann et al. (2020) suggested that their preclinical results supported the hypothesis that individuals at high risk of developing AML might benefit from targeted epigenetic therapy in a preventative setting.
In a mouse model with a hematopoietic-specific inactivation of Dnmt3a, Kim et al. (2021) found reduced trabecular bone volume. Histologic studies in the mutant mice demonstrated an increased number of osteoclasts and a normal number of osteoblasts on bone surfaces compared to wildtype mice. In mice with a bone marrow-specific heterozygous R878H mutation in Dnmt3a, Kim et al. (2021) also observed increased osteoclasts and normal number of osteoblasts on bone surfaces compared to wildtype mice. These results suggested that effects on bone mass caused by Dnmt3a hematopoietic mutations were mediated by osteoclasts. Analysis of myeloid and osteoclast cells from the Dnmt3a-mutant mice in culture showed that the myeloid cells secrete proinflammatory cytokines that increase osteoclast differentiation. Alendronate treatment improved the decreased cortical bone area and femoral bending strength in the bone marrow-specific heterozygous Dnmt3a R878H mutant mice.
Dura et al. (2022) found that Dnmt3a -/- mice were developmentally delayed after birth and died around 25 days postpartum. Dnmt3a -/- males showed significant and increasing reduction in testis weight and seminiferous tubule surface compared with wildtype. Whole-genome bisulfite sequencing (WGBS) on DNA from mouse embryonic prospermatogonia showed that Dnmt3a largely indiscriminately methylated the whole genome in prenatal male germ cells. However, Dnmt3a did not methylate young retrotransposon promoters, and therefore it did not silence retrotransposons during spermatogenesis. Analysis of testes of mice with conditional Dnmt3a deletion at different ages revealed that mutant mice displayed progressive loss of spermatogenic ability, as spermatogenesis only progressed through the first wave, after which spermatogenic potential was lost. Despite progressive spermatogenic decline, spermatogonial stem cell (SSCs) were specified, self-renewed, and maintained their most naive state, although excessively. However, Dnmt3a -/- SSCs were not able to exit the stem cell state in the absence of Dnmt3a-dependent DNA methylation, resulting in spermatogenic failure. Further analysis revealed that DNA methylation regulated SSC plasticity, and that lack of Dnmt3a-dependent DNA methylation was associated with ectopic recruitment of enhancers in SSCs, which activated or enhanced expression of genes that sustained SSC identity.
Tovy et al. (2022) found that Dnmt3a +/- mice were indistinguishable from wildtype until around 3 months of age. Dnmt3a +/- mice developed adipose tissue expansion and weight gain, recapitulating the human TBRS phenotype. Moreover, Dnmt3a +/- mice showed increased food intake and decreased energy expenditure, leading to the development of glucose and insulin resistance. Adipose tissue in Dnmt3a +/- mice exhibited defects in preadipocyte maturation and precocious activation of inflammatory gene networks, including interleukin-6 (IL6; 605509) signaling. Adipocyte progenitor cell lines lacking Dnmt3a also exhibited aberrant differentiation. Mice with conditional deletion of Dnmt3a in adipocyte progenitors showed enlarged fat depots and increased progenitor numbers, partly recapitulating the TBRS obesity phenotype. Loss of Dnmt3a led to constitutive DNA hypomethylation, such that the DNA methylation landscape of young adipocyte progenitors resembled that of older wildtype mice.
Gu et al. (2022) found that Dnmt3a1 -/- mice were runted, with smaller spleen and thymus than wildtype, and died 3 to 4 weeks after birth. In contrast, Dnmt3a2 -/- mice displayed no phenotype, were fertile, and had a normal lifespan. The findings suggested that Dnmt3a1 is the predominant isoform in postnatal tissues and that Dnmt3a1, but not Dnmt3a2, is essential for postnatal development. Dnmt3a1 was exclusively and highly expressed in all postnatal brain regions, and it bound to and regulated bivalent neurodevelopmental genes in cerebral cortex. Moreover, the authors generated mice with conditional Dnmt3a1 deletion and found that Dnmt3a1 restoration in nervous system largely rescued the runting phenotype and early postnatal lethality of Dnmt3a2 -/- mice. Compared with Dnmt3a2, Dnmt3a1 has an additional 219 amino acids at its N terminus, which is intrinsically disordered. Functionally, the N terminus facilitated Dnmt3a1 enrichment at bivalent promoters of neurodevelopmental genes by recognizing and binding to the monoubiquitinated histone H2AK119 in nucleosomes.
In a 3-year-old patient with Tatton-Brown-Rahman syndrome (TBRS; 615879), Tatton-Brown et al. (2014) identified a de novo heterozygous 3-bp deletion (c.889_891delTGG) in the DNMT3A gene, resulting in the deletion of Trp297 in the PWWP domain. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not present in 1,000 control exomes. In vitro functional studies of the variant were not performed.
In a 19-year-old patient with Tatton-Brown-Rahman syndrome (TBRS; 615879), Tatton-Brown et al. (2014) identified a de novo heterozygous c.1943T-C transition in the DNMT3A gene, resulting in a leu648-to-pro (L648P) substitution in the methyltransferase domain. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not present in 1,000 control exomes. In vitro functional studies of the variant were not performed.
In a 9-year-old patient with Tatton-Brown-Rahman syndrome (TBRS; 615879), Tatton-Brown et al. (2014) identified a de novo heterozygous c.929T-A transversion in the DNMT3A gene, resulting in an ile310-to-asn (I310N) substitution in the PWWP domain. In vitro functional studies of the variant were not performed.
In a 4-year-old patient with Tatton-Brown-Rahman syndrome (TBRS; 615879), Tatton-Brown et al. (2014) identified a de novo heterozygous c.1643T-A transversion in the DNMT3A gene, resulting in a met548-to-lys (M548K) substitution in the zinc finger ADD domain. In vitro functional studies of the variant were not performed.
In a 9.8-year-old patient with Tatton-Brown-Rahman syndrome (TBRS; 615879), Tatton-Brown et al. (2014) identified a de novo heterozygous c.2705T-C transition in the DNMT3A gene, resulting in a phe902-to-ser (F902S) substitution in the methyltransferase domain. In vitro functional studies of the variant were not performed.
Tatton-Brown-Rahman Syndrome
In 2 unrelated patients with Tatton-Brown-Rahman syndrome (TBRS; 615879), Shen et al. (2017) identified a de novo heterozygous c.2645G-A transition (c.2645G-A, NM_175629.2) in the DNMT3A gene, resulting in an R882H substitution.
In a 6-year-old girl with TBRS, Kosaki et al. (2017) identified heterozygosity for the de novo R882H mutation in the DNMT3A gene.
Acute Myeloid Leukemia, Somatic
Of 62 patients with acute myelogenous leukemia (AML; 601626) who were found to have a somatic mutation in the DNMT3A gene, Ley et al. (2010) found that 27 had a C-to-T transition at a CpG dinucleotide, resulting in an arg882-to-his (R882H) substitution.
Functional Studies of DNMT3A R882H
Using size-exclusion chromatography, Nguyen et al. (2019) confirmed that human DNMT3A formed large oligomeric species, as well as smaller complexes around the size of a tetramer, with large oligomers having lower methyltransferase activity relative to smaller complexes. The dominant-negative DNMT3A R882H mutant stabilized DNMT3A oligomer formation and shifted the DNMT3A oligomer equilibrium toward higher-order multimers, resulting in a dose-dependent reduction of enzyme activity compared with wildtype DNMT3A. In contrast, mutations that disrupted the oligomer-forming interface of the DNMT3A catalytic domain caused a significant shift from large oligomers to smaller species, with reduced enzymatic activity relative to wildtype and comparable with that of R882H. DNMT3L (606588) disrupted formation of large oligomers to activate wildtype DNMT3A by binding to and breaking down higher-order DNMT3A into smaller, more active complexes. Likewise, DNMT3L bound to the R882H mutant, but activity of the R882H mutant was only partially restored relative to wildtype DNMT3A, suggesting that R882H interferes with DNMT3A methyltransferase activity by an additional mechanism. Further analysis revealed that the R882H mutation also compromised the DNA-binding ability of DNMT3A.
Sandoval and Reich (2019) found that wildtype DNMT3A and the DNMT3A R882H mutant were differentially responsive to modulation by p53 (TP53; 191170), as p53 failed to inhibit methylation activity of DNMT3A R882H.
Using purified recombinant proteins, Norvil et al. (2020) showed that DNMT3A R882 mutants lost the cooperative kinetic mechanism in methylation of DNA substrates compared with wildtype DNMT3A. R882 played a key role in the interaction of DNMT3A with DNA, and the R882H mutation altered the specificity of DNMT3A such that it adopted a substrate preference similar to that of DNMT3B (602900), making DNMT3A R882H a DNMT3B-like enzyme. The authors noted that DNMT3A and DNMT3B redundantly methylate many genomic regions in cells, but they also have preferred and specific targets, as Dnmt3a preferentially methylates major satellite repeats in pericentric regions in mouse cells, whereas Dnmt3b preferentially methylates minor satellite repeats in centromeric regions. Analysis with mouse embryonic stem cells revealed that mouse Dnmt3a R878H mutant retained activity for minor satellite DNA and methylated Dnmt3b-preferred target sites but lost its preference for sites methylated by Dnmt3a.
Tatton-Brown-Rahman Syndrome
In a patient with Tatton-Brown-Rahman syndrome (TBRS; 615879), Shen et al. (2017) identified a c.2644C-T transition (c.2644C-T, NM_175629.2) in the DNMT3A gene, resulting in an arg882-to-cys (R882C) substitution.
In an 19-year-old Dutch boy with TBRS who developed FAB type M5 acute myelogenous leukemia (AML; 601626) at age 15 years, Hollink et al. (2017) identified a de novo heterozygous germline c.2644C-T transition in the DNMT3A gene, resulting in an arg882-to-cys (R882C) substitution. The mutation was found by whole-exome sequencing and confirmed by Sanger sequencing. Analysis of leukemic cells showed an aberrant karyotype and a somatic PTPN11 mutation (T73I; 176876.0011), but no additional somatic mutations or loss of heterozygosity of the DNMT3A gene. Hollink et al. (2017) suggested that mutations at the R882 residue, which are associated with acute myeloid leukemia in the somatic state, may also predispose germline carriers of the mutation to the development of AML. The authors postulated epigenetic dysregulation as a possible molecular mechanism and suggested that patients with TBRS be followed for hematologic malignancies. Functional studies of the variants were not performed.
In a 6-year-old girl (patient 5) with TBRS, Balci et al. (2020) identified heterozygosity for the recurrent R882C mutation in the DNMT3A gene. The child was previously reported by DeMari et al. (2016) at age 3.5 years with a heterozygous frameshift mutation in the CLTC gene (118955.0001) and a diagnosis of global developmental delay (MRD56; 617854). At age 6 years, the child presented with lymphadenopathy, a mediastinal mass, and hypercalcemia, and was diagnosed with T-cell lymphoblastic lymphoma.
Acute Myeloid Leukemia, Somatic
Of 62 patients with acute myelogenous leukemia (AML; 601626) who were found to have a somatic mutation in the DNMT3A gene, Ley et al. (2010) found that 7 had a C-to-T transition at a CpG dinucleotide, resulting in an arg882-to-cys (R882C) substitution.
In 2 unrelated children (patients 1 and 2) with Heyn-Sproul-Jackson syndrome (HESJAS; 618724), Heyn et al. (2019) identified a de novo heterozygous c.988T-C transition (c.988T-C, NM_175629.2) in the DNMT3A gene, resulting in a trp330-to-arg (W330R) substitution at a highly conserved residue in the PWWP domain. The mutation, which was found by whole-exome or next-generation sequencing and confirmed by Sanger sequencing, was not found in the gnomAD database. Detailed in vitro and in vivo functional expression studies in patient-derived fibroblasts and peripheral leukocytes showed abnormal hypermethylation at various polycomb repression-associated regions, resulting in aberrant expression of genes involved in developmental processes. The findings were consistent with a gain-of-function effect.
In a 4-year-old boy of Spanish descent (patient 3) with Heyn-Sproul-Jackson syndrome (HESJAS; 618724), Heyn et al. (2019) identified a de novo heterozygous c.997G-A transition (c.997G-A, NM_175629.2) in the DNMT3A gene, resulting in an asp333-to-asn (D333N) substitution at a conserved residue in the PWWP domain. The mutation, which was found by trio-based exome sequencing and confirmed by Sanger sequencing, was not found in the 1000 Genomes Project or ExAC databases. Detailed in vitro and in vivo functional expression studies in patient-derived fibroblasts and peripheral leukocytes showed abnormal hypermethylation at various polycomb repression-associated regions, resulting in aberrant expression of genes involved in developmental processes. The findings were consistent with a gain-of-function effect.
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