DNM1 Gene and Its Related Epileptic Phenotypes

AbstractThe phenotypic variety associated to mutations in dynamin 1 (DNM1), codifying the presynaptic protein DNM1 has been increasingly reported, mainly related to encephalopathy with intractable epilepsy; currently, it is known the phenotype related to DNM1 gene mutations is relatively homogeneous with developmental delay, hypotonia, and epilepsy characterized by infantile spasms and possible progression to Lennox-Gastaut syndrome. By examining all the papers published until 2020 (18 articles), we compared data from 30 patients (extrapolated from 5 papers) with DNM1 mutations, identifying 26 patients with de novo mutations in DNM1. Nine patients (33.3%) reported the recurrent mutation p.Arg237Trp. A usual phenotype observed comprises severe to deep developmental delay and muscular hypotonia in all patients with epilepsy beginning with infantile spasms, which often evolved into Lennox-Gastaut syndrome. Data about GTPase or central domains mutations, and existing structural modeling and functional suggest a dominant negative effect on DMN1 function. Generally genetic epilepsies consist of a wide spectrum of clinical features, unlike that, DNM1-related CNS impairment phenotype is quite uniform. In up to one third of patients it has been found variant p.Arg237Trp, which is one of the most frequent variant detected in epileptic encephalopathies. The understanding of DNM1 function opens up the chance that this gene would become a new therapeutic target for epilepsies.

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Biallelic and monoallelic variants in PLXNA1 are implicated in a novel neurodevelopmental disorder with variable cerebral and eye anomalies

Genetics in Medicine ◽

10.1038/s41436-021-01196-9 ◽

2021 ◽

Author(s):

Gabriel C. Dworschak ◽

Jaya Punetha ◽

Jeshurun C. Kalanithy ◽

Enrico Mingardo ◽

Haktan B. Erdem ◽

...

Keyword(s):

Developmental Delay ◽

De Novo ◽

Neurodevelopmental Disorder ◽

Structural Modeling ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Global Developmental Delay ◽

Downstream Signaling ◽

Negative Effect

Abstract Purpose To investigate the effect of PLXNA1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and to functionally characterize the zebrafish homologs plxna1a and plxna1b during development. Methods We assembled ten patients from seven families with biallelic or de novo PLXNA1 variants. We describe genotype–phenotype correlations, investigated the variants by structural modeling, and used Morpholino knockdown experiments in zebrafish to characterize the embryonic role of plxna1a and plxna1b. Results Shared phenotypic features among patients include global developmental delay (9/10), brain anomalies (6/10), and eye anomalies (7/10). Notably, seizures were predominantly reported in patients with monoallelic variants. Structural modeling of missense variants in PLXNA1 suggests distortion in the native protein. Our zebrafish studies enforce an embryonic role of plxna1a and plxna1b in the development of the central nervous system and the eye. Conclusion We propose that different biallelic and monoallelic variants in PLXNA1 result in a novel neurodevelopmental syndrome mainly comprising developmental delay, brain, and eye anomalies. We hypothesize that biallelic variants in the extracellular Plexin-A1 domains lead to impaired dimerization or lack of receptor molecules, whereas monoallelic variants in the intracellular Plexin-A1 domains might impair downstream signaling through a dominant-negative effect.

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An ensemble of toxic channel types underlies the severity of the de novo variant G375R of the human BK channel

10.1101/2021.12.22.473917 ◽

2021 ◽

Author(s):

Yanyan Geng ◽

Ping Li ◽

Alice Butler ◽

Bill Wang ◽

Lawrence Salkoff ◽

...

Keyword(s):

Neuronal Excitability ◽

De Novo ◽

Bk Channels ◽

Bk Channel ◽

Dominant Negative ◽

Dominant Negative Effect ◽

De Novo Mutations ◽

Negative Effect ◽

De Novo Variant ◽

Heteromeric Channels

De novo mutations play a prominent role in neurodevelopmental diseases including autism, schizophrenia, and intellectual disability. Many de novo mutations are dominant and so severe that the afflicted individuals do not reproduce, so the mutations are not passed into the general population. For multimeric proteins, such severity may result from a dominant-negative effect where mutant subunits assemble with WT to produce channels with adverse properties. Here we study the de novo variant G375R heterozygous with the WT allele for the large conductance voltage- and Ca2+-activated potassium (BK) channel, Slo1. This variant has been reported to produce devastating neurodevelopmental disorders in three unrelated children. If mutant and WT subunits assemble randomly to form tetrameric BK channels, then ~6% of the assembled channels would be wild type (WT), ~88% would be heteromeric incorporating from 1-3 mutant subunits per channel, and ~6% would be homomeric mutant channels consisting of four mutant subunits. To test this hypothesis, we analyzed the biophysical properties of single BK channels in the ensemble of channels expressed following a 1:1 injection of mutant and WT cRNA into oocytes. We found ~3% were WT channels, ~85% were heteromeric channels, and ~12% were homomeric mutant channels. All of the heteromeric channels as well as the homomeric mutant channels displayed toxic properties, indicating a dominant negative effect of the mutant subunits. The toxic channels were open at inappropriate negative voltages, even in the absence of Ca2+, which would lead to altered cellular function and decreased neuronal excitability.

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A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect

The American Journal of Human Genetics ◽

10.1016/j.ajhg.2016.03.018 ◽

2016 ◽

Vol 98 (5) ◽

pp. 981-992 ◽

Cited By ~ 44

Author(s):

Meriel McEntagart ◽

Kathleen A. Williamson ◽

Jacqueline K. Rainger ◽

Ann Wheeler ◽

Anne Seawright ◽

...

Keyword(s):

De Novo ◽

Dominant Negative ◽

Dominant Negative Effect ◽

De Novo Mutations ◽

Negative Effect

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DNM1 encephalopathy

Neurology ◽

10.1212/wnl.0000000000004152 ◽

2017 ◽

Vol 89 (4) ◽

pp. 385-394 ◽

Cited By ~ 31

Author(s):

Sarah von Spiczak ◽

Katherine L. Helbig ◽

Deepali N. Shinde ◽

Robert Huether ◽

Manuela Pendziwiat ◽

...

Keyword(s):

Functional Data ◽

De Novo ◽

Structural Modeling ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Global Developmental Delay ◽

Epilepsy Syndrome ◽

De Novo Mutations ◽

Phenotypic Data ◽

Phenotypic Spectrum

Objective:To evaluate the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling.Methods:We reviewed phenotypic data of 21 patients (7 previously published) with DNM1 mutations. We compared mutation data to known functional data and undertook biomolecular modeling to assess the effect of the mutations on protein function.Results:We identified 19 patients with de novo mutations in DNM1 and a sibling pair who had an inherited mutation from a mosaic parent. Seven patients (33.3%) carried the recurrent p.Arg237Trp mutation. A common phenotype emerged that included severe to profound intellectual disability and muscular hypotonia in all patients and an epilepsy characterized by infantile spasms in 16 of 21 patients, frequently evolving into Lennox-Gastaut syndrome. Two patients had profound global developmental delay without seizures. In addition, we describe a single patient with normal development before the onset of a catastrophic epilepsy, consistent with febrile infection-related epilepsy syndrome at 4 years. All mutations cluster within the GTPase or middle domains, and structural modeling and existing functional data suggest a dominant-negative effect on DMN1 function.Conclusions:The phenotypic spectrum of DNM1-related encephalopathy is relatively homogeneous, in contrast to many other genetic epilepsies. Up to one-third of patients carry the recurrent p.Arg237Trp variant, which is now one of the most common recurrent variants in epileptic encephalopathies identified to date. Given the predicted dominant-negative mechanism of this mutation, this variant presents a prime target for therapeutic intervention.

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De novo Mutations in NALCN Cause a Syndrome of Congenital Contractures of the Limbs and Face with Hypotonia, and Developmental Delay

10.1101/013656 ◽

2015 ◽

Author(s):

Jessica X Chong ◽

Margaret J McMillin ◽

Kathryn M Shively ◽

Anita E Beck ◽

Colby T Marvin ◽

...

Keyword(s):

Exome Sequencing ◽

Developmental Delay ◽

De Novo ◽

Dominant Negative ◽

Protein Domain ◽

Dominant Negative Effect ◽

Global Developmental Delay ◽

Missense Mutations ◽

Severe Intellectual Disability ◽

Dominant Condition

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five simplex cases putatively diagnosed with “DA2A with severe neurological abnormalities” in which the proband had Congenital Contractures of the LImbs and FAce, Hypotonia, and global Developmental Delay often resulting in early death, a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in sodium leak channel, nonselective (NALCN) in four families with CLIFAHDD syndrome. Using molecular inversion probes to screen NALCN in a cohort of 202 DA cases as well as concurrent exome sequencing of six other DA cases revealed NALCN mutations in ten additional families with “atypical” forms of DA. All fourteen mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that mutant NALCN nearly abolished the expression of wildtype NALCN, suggesting that mutations that cause CLIFAHDD syndrome have a dominant negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families with an autosomal recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition with varied though overlapping phenotypic features perhaps depending on the type of mutation and affected protein domain(s).

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Four novel mutations in the ALPL gene in Chinese patients with odonto, childhood, and adult hypophosphatasia

Bioscience Reports ◽

10.1042/bsr20171377 ◽

2018 ◽

Vol 38 (4) ◽

Cited By ~ 2

Author(s):

Lijun Xu ◽

Qianqian Pang ◽

Yan Jiang ◽

Ou Wang ◽

Mei Li ◽

...

Keyword(s):

Serum Alkaline Phosphatase ◽

Gene Mutations ◽

Dominant Negative ◽

Chinese Patients ◽

Dominant Negative Effect ◽

Healthy Controls ◽

Missense Mutations ◽

Genetic Characteristics ◽

Chinese Families ◽

Negative Effect

Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone and/or dental mineralization, and decreased serum alkaline phosphatase (ALP) activity. ALPL, the only gene related with HPP, encodes tissue non-specific ALP (TNSALP). Few studies were carried out in ALPL gene mutations in the Chinese population with HPP. The purpose of the present study is to elucidate the clinical and genetic characteristics of HPP in five unrelated Chinese families and two sporadic patients. Ten clinically diagnosed HPP patients from five unrelated Chinese families and two sporadic patients and fifty healthy controls were genetically investigated. All 12 exons and exon–intron boundaries of the ALPL gene were amplified by PCR and directly sequenced. The laboratory and radiological investigations were conducted simultaneously in these HPP ten patients. A 3D model of the TNSALP was used to predict the dominant negative effect of identified missense mutations. Three odonto, three childhood, and four adult types of HPP were clinically diagnosed. Ten mutations were identified in five unrelated Chinese families and two sporadic patients, including eight missense mutations and two frameshift mutations. Of which, four were novel: one frameshift mutation (p.R138Pfsx45); three missense mutations (p.C201R, p.V459A, p.C497S). No identical mutations and any other new ALPL mutations were found in unrelated 50 healthy controls. Our study demonstrated that the ALPL gene mutations are responsible for HPP in these Chinese families. These findings will be useful for clinicians to improve understanding of this heritable bone disorder.

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Characterization of ANGPT2 mutations associated with primary lymphedema

Science Translational Medicine ◽

10.1126/scitranslmed.aax8013 ◽

2020 ◽

Vol 12 (560) ◽

pp. eaax8013 ◽

Cited By ~ 1

Author(s):

Veli-Matti Leppänen ◽

Pascal Brouillard ◽

Emilia A. Korhonen ◽

Tuomas Sipilä ◽

Sawan Kumar Jha ◽

...

Keyword(s):

De Novo ◽

Vascular System ◽

Lymphatic Vessels ◽

Mouse Skin ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Missense Mutations ◽

Activation Mechanisms ◽

Primary Lymphedema ◽

Negative Effect

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor–like domains 1 and 2) receptor complex and α5β1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)–ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

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239-kb Microdeletion Spanning KMT2E in a Child with Developmental Delay: Further Delineation of the Phenotype

Molecular Syndromology ◽

10.1159/000516635 ◽

2021 ◽

pp. 1-6

Author(s):

Konstantina Kosma ◽

Konstantinos Varvagiannis ◽

Anastasios Mitrakos ◽

Maria Tsipi ◽

Joanne Traeger-Synodinos ◽

...

Keyword(s):

Developmental Delay ◽

De Novo ◽

Neurodevelopmental Disorder ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Chromosomal Microarray ◽

Global Developmental Delay ◽

Common Mechanism ◽

Facial Dysmorphism ◽

Gi Symptoms

Pathogenic KMT2E variants underly O'Donnell-Luria-Rodan syndrome, a recently described neurodevelopmental disorder characterized by global developmental delay, variable degrees of intellectual disability, and subtle facial dysmorphism. Less common findings include autism, seizures, gastrointestinal (GI) problems, and abnormal head circumference. Occurrence of mostly truncating variants as well as the similar phenotype observed in individuals with deletions spanning KMT2E suggest haploinsufficiency of this gene as a common mechanism for the disorder, while a gain-of-function or dominant-negative effect cannot be ruled out for some missense variants. Deletions reported in the literature encompass several additional known or presumed haploinsufficient genes, thus leading to more complex phenotypes. Here, we describe a male with antenatal onset hydronephrosis, hypotonia, global developmental delay, prominent GI symptoms as well as facial dysmorphism. Chromosomal microarray revealed a 239-kb de novo microdeletion spanning KMT2E and LHFPL3. Clinical presentation of our proband, harboring one of the smallest deletions of the region confirms the core features of this disorder, suggests GI symptoms as a prominent finding in affected individuals while expanding the phenotypic spectrum to abnormalities of the urinary tract.

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Mutant p53 Enhances the Development and Sustained Growth of MYC-Driven Lymphoma and Exerts a Dominant Negative Effect Preferentially Deregulating Pathways for Metabolism and DNA Repair

Blood ◽

10.1182/blood.v128.22.1545.1545 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1545-1545

Author(s):

Brandon J Aubrey ◽

Ana Janic ◽

Yunshun Chen ◽

Gordon K Smyth ◽

Andrew J Kueh ◽

...

Keyword(s):

Dna Damage ◽

Target Genes ◽

Human Cancer ◽

Gene Mutations ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Wild Type ◽

Over Expression ◽

Negative Effect ◽

Induced Apoptosis

Abstract Over-expression of the c-MYC oncogene and Trp53 gene mutations are among the most common genetic alterations in human cancer and, when combined, result in highly aggressive malignant disease. Trp53 gene mutations produce over-expressed mutant TRP53 proteins that drive cancer growth through both loss of wild-type Trp53 tumor suppressor function and gain-of-function oncogenic properties. The Eμ-Myc mouse model provides a setting to study the functional interplay between c-Myc over-expression and mutant TRP53 proteins. Eμ-Myc transgenic mice carry a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma, and develop aggressive pre-B/B cell lymphoma with a high (~20%) spontaneous rate of Trp53 mutation. The effect of five mouse mutant TRP53 proteins (V170M, I192S, G280, R246Q, R270H) was initially examined in three settings (Trp53-/-, Trp53+/- and Trp53+/+;Eμ-Myc) using a hematopoietic stem and progenitor cell (HSPC) reconstitution model. Each mutant TRP53 protein studied corresponds to a commonly re-occurring Tp53 mutation in human cancer. Retroviral over-expression enabled the comparison of mutant-specific and genotype-specific features for each mutant TRP53 protein. Mutant TRP53 expression did not accelerate lymphoma development in mice receiving Trp53-/- or Trp53+/- HSPCs. However, mice reconstituted with Trp53+/- HSPCs expressing the TRP53 mutants displayed an altered tumor spectrum compared to mice reconstituted with control Trp53+/- HSPCs. In contrast, mutant TRP53 markedly accelerated lymphoma development in mice receiving Trp53+/+;Eμ-Myc HSPCs, highlighting a synergy between c-Myc over-expression and Trp53 mutations in neoplastic transformation. Furthermore, inducible mutant TRP53 expression demonstrated a dependency on sustained expression of mutant TRP53 in established MYC-driven lymphomas. Notably, none of the c-MYC plus mutant TRP53 driven lymphomas exhibited spontaneous endogenous Trp53 mutations. Despite the enhanced tumorigenesis, most established lymphomas from this model displayed sensitivity to TRP53-activating drugs consistent with a weak dominant negative effect over wild-type Trp53-induced apoptosis. Consistent with this finding, pre-malignant Trp53+/+;Eμ-Myc primary B-cells expressing mutant TRP53 were not protected against Trp53-induced apoptosis. Pre-malignant B-cells displayed a small increase in cell cycling and an expansion of the tumor-initiating pre/pro-B cell population. Most significantly, functional assessment of DNA damage in pre-malignant cells, using single cell gel electrophoresis (comet assay) and γ-H2AX staining, revealed increased DNA damage, suggesting an important role for defects in DNA repair during mutant TRP53-driven lymphoma development. To investigate the nature of the dominant negative effect, mutant TRP53 protein was exogenously expressed in mouse Eµ-Myc Trp53+/+ lymphoma cell lines. The impact of mutant TRP53 on the transcriptional function of the endogenous wild-type TRP53 protein was then studied using the TRP53-activating compound, nutlin-3a. Surprisingly, in established lymphoma cell lines, mutant TRP53 impaired nutlin-3a-induced apoptosis despite substantial induction of the critical pro-apoptotic effector, PUMA. To explore this finding further, we globally characterized the dominant negative effect, and assessed for mutant TRP53-specific transcriptional targets, by performing whole transcriptome sequence (RNAseq) analysis after treatment with nutlin-3a. Analysis of known wild-type Trp53 target genes (n=283) demonstrated that the induction of these genes as a group was repressed in the presence of the mutant TRP53 protein (ROAST test, p=6.7e-04). Remarkably, however, mutant TRP53 significantly repressed only 57% of the nutlin-3a-induced Trp53 target genes. Analysis of these strongly repressed genes highlighted the importance of several pathways, including metabolism, DNA damage repair and negative feedback loops in TRP53 signaling. This suggests a previously unrecognized selectivity of the dominant-negative-effect for certain p53 pathways that may be important in cancer initiation. Additional mutant TRP53-specific transcriptional targets were also identified and are under further investigation. Disclosures No relevant conflicts of interest to declare.

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R882H DNMT3A Causes Dominant-Negative Inhibition Of WT DNMT3A

Blood ◽

10.1182/blood.v122.21.3812.3812 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3812-3812

Author(s):

David A. Russler-Germain ◽

David H Spencer ◽

Margaret A. Young ◽

Tamara Lamprecht ◽

Chris Miller ◽

...

Keyword(s):

Dna Methylation ◽

Catalytic Domain ◽

De Novo ◽

Dominant Negative ◽

Size Exclusion ◽

Dominant Negative Effect ◽

Methyltransferase Activity ◽

Negative Effect

Abstract Mutations in DNMT3A (encoding one of two mammalian de novo DNA methyltransferases) are found in >30% of normal karyotype AML cases and correlate with poor clinical outcomes. Most DNMT3A mutations occur at position R882 within the catalytic domain (most commonly R882H) and are virtually always heterozygous. This over-representation suggests that mutations at R882 may result in gain-of-function or dominant-negative activity that contributes to leukemogenesis. However, how DNA methylation might be altered in DNMT3A-mutant cases of AML remains unclear, and no published study to date has addressed the effects of mixing wild-type (WT) and R882H DNMT3A. Importantly, mouse HSPCs deficient in Dnmt3a dramatically expand over time and have a concurrent defect in differentiation (Challen, GA et al. Nat Genet, 2011). Mice haploinsufficient for Dnmt3a, on the other hand, do not have a measurable defect in hematopoiesis. Collectively, these data suggest that the heterozygous R882 mutations probably cause more than a simple loss-of-function phenotype. We purified full-length, human WT and R882H DNMT3A using a mammalian tissue culture system to produce recombinant proteins for biochemical modeling of the de novo methylation potential of a DNMT3A-mutant AML cell. rhR882H DNMT3A exhibits roughly 10-20% of the de novo DNA methyltransferase activity of rhWT DNMT3A, similar to observations by other groups. We added increasing amounts of R882H DNMT3A to a fixed amount of WT DNMT3A and observed a linear increase in the net enzymatic activity, reflecting the summed activity of the two forms of DNMT3A in these 4-hour in vitro reactions. In contrast, 12-hour in vitro DNA methylation assays with mixed WT and R882H DNMT3A demonstrated net methylation less than the predicted summed activity of the two enzymes, suggesting that a dominant-negative effect of R882H DNMT3A may occur with a long equilibration time. To better simulate an AML cell with a heterozygous R882H mutation, we co-transfected HEK293T cells with equal amounts of poly-His-tagged WT and R882H DNMT3A expression vectors. Subsequently co-purified (i.e. in vivo-mixed) WT and R882H DNMT3A exhibited a striking reduction in methyltransferase activity, with total activity similar to R882H DNMT3A alone (Figure 1A). TSQ mass spectrometry allowed us to verify the presence and quantify the relative concentration of WT and R882H DNMT3A in our co-purified samples. We exploited a novel tryptic cleavage site in DNMT3A produced by the R882H mutation to generate standard concentration curves using recombinant peptides distinguishing the two protein forms. Our co-purified enzyme preparations had WT:R882H ratios ranging from 0.79 to 1.60; all demonstrated the dominant-negative effect of R882H. DNMT3A is a processive enzyme, catalyzing multiple methyl-group transfers before dissociating from target DNA. This is dependent on the ability of WT DNMT3A to form homo-oligomers (tetramers and larger), which was recently shown to be disrupted by the R882H mutation using the catalytic domain of DNMT3A produced in E.coli (Holz-Schietinger, C et al. JBC, 2012). We therefore postulated that the dominant-negative effect of R882H may be due to the disruption of WT DNMT3A oligomerization. Using a Superose 6 size exclusion column, we confirmed the tetramerization defect of R882H DNMT3A relative to WT DNMT3A. Notably, in vivo-mixed (co-purified) WT and R882H DNMT3A complexes exhibited a pattern of oligomerization identical to R882H DNMT3A alone. However, WT and R882H DNMT3A mixed in vitro exhibited a distribution of oligomers corresponding to the expected average of the WT and R882H curves (Figure 1B). These data demonstrate that production of equal amounts of WT and R882H DNMT3A within the same cell provides an environment where R882H DNMT3A can exert a potent dominant-negative effect on WT DNMT3A. Furthermore, our data suggest that this effect is associated with diminished formation of tetramers when WT and R882H DNMT3A are complexed together. Thus, the R882H mutation has two distinct consequences that affect DNMT3A activity in AML cells: 1) it severely reduces its own de novo methyltransferase activity, and 2) it disrupts the ability of WT DNMT3A to form functional tetramers. These two effects severely reduce total DNMT3A activity in AML cells, and may explain why this mutation is virtually always heterozygous in AML samples, since homozygosity would not further reduce DNMT3A activity. Disclosures: No relevant conflicts of interest to declare.

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