De Novo Mutations in EBF3 Cause a Neurodevelopmental Syndrome

Abstract

Early B cell factor 3 (EBF3) is an atypical transcription factor that is thought to influence the laminar formation of the cerebral cortex. Here, we report that de novo mutations in EBF3 cause a complex neurodevelopmental syndrome. The mutations were identified in two large-scale sequencing projects: the UK Deciphering Developmental Disorders (DDD) study and the Canadian Clinical Assessment of the Utility of Sequencing and Evaluation as a Service (CAUSES) study. The core phenotype includes moderate to severe intellectual disability, and many individuals exhibit cerebellar ataxia, subtle facial dysmorphism, strabismus, and vesicoureteric reflux, suggesting that EBF3 has a widespread developmental role. Pathogenic de novo variants identified in EBF3 include multiple loss-of-function and missense mutations. Structural modeling suggested that the missense mutations affect DNA binding. Functional analysis of mutant proteins with missense substitutions revealed reduced transcriptional activities and abilities to form heterodimers with wild-type EBF3. We conclude that EBF3, a transcription factor previously unknown to be associated with human disease, is important for brain and other organ development and warrants further investigation.

Figure 1

PubMed Score versus Cadd_sum Score Shown are x-y plots to prioritize genes associated with ataxia. The Cadd_sum score is on the x axis, the PubMed score is on the y axis, and the Loess regression line is shown in blue. Known genes are highlighted in yellow, and a selected few known ataxia-related genes are labeled with gene symbols. EBF3 is shaded red. Only de novo variants are presented in this analysis. The PubMed score was calculated as follows: all genes on the variant list were searched in PubMed for keywords “[gene name] AND (ataxia [title/abstract] OR cerebellar [title/abstract] OR cerebellum [Title/Abstract] OR cortex [title/abstract] OR intellectual [title/abstract] OR neuron [title/abstract]).” The returned top 50 articles were searched for the occurrence of the searched keywords (ataxia, cerebellar, cerebellum, cortex, intellectual, and neuron), and the PubMed score was calculated as the sum of the occurrence. The assumption is that the PubMed score captures the relevance between the phenotype and a given gene as supported by the literature. The Cadd_sum was calculated as follows: all variants on the list were assigned a CADD score through the CADD web service. For de novo variants, only those where an alternative allele was called in the child but in neither parent were counted (the collection is denoted as denovo_v), and the Cadd_sum of a given gene was calculated on a max per-individual level. (We believe only the most damaging de novo variant of a given gene contributes to the observed phenotype. In fact, for any individual in the cohort, no two or more de novo variants were found on the same gene.) If we have M individuals [1… m … M], G genes [1 … g … G], and V variants [1…v… V], the Cadd_sum of a gene g for de novo cases is given by the following formula: ${\sum }_{\text{m}}^{\text{M}}\left\{{\text{Max}}_{\text{v}\in \text{g},\text{v}\in \text{m},\text{v}\in \text{de}\text{novo}\_\text{v}}\left(\text{CADD}\left(\text{v}\right)\right)\right\}.$. The method has been validated with other keyword searches (Figure S1) and demonstrates that for Cadd_sum scores greater than 10, there is a linear increase in the PubMed score. The correlation between the PubMed score and the Cadd_sum score, which is observed only when relevant phenotype terms are used in the PubMed search, as well as the successful annotation of known ataxia-related genes with high specificity, demonstrates the method’s efficacy in the discovery of pathogenic genes in the cohort.

Figure 2

Schematic Representations of EBF3 and EBF3 Structure (A) Structure of EBF3, including the position of the zinc knuckle within the DBD. Numbering refers to amino acids. (B) Exon structure of EBF3. Numbering at the top refers to amino acid residues. Mutations identified in this study are shown in red with arrows. (C) Structure of mouse EBF1 (PDB: 3MLP) in complex with DNA (the dimer chain is hidden for clarity); it was created with CCP4mg. Mouse EBF1 has 89% sequence identity with human EBF3 and 100% in the Zinc knuckle. The protein is shown as a blue-gray ribbon, and the DNA is green. The five EBF3 missense variants are labeled and displayed as space-filling models. (D) Close up of DNA-binding interactions of EBF3 missense variants (generated with PyMol). The protein is shown in green, and the DNA is shown in orange and magenta. Depicted are the numerous interactions involving Pro177 and Lys193.

Figure 3

Dose-Dependent Effects of EBF3 on Function Flow cytometric analysis of mIgM on plasmacytoma cells in response to increasing amounts of wild-type or mutant EBF3 (each by itself). The retroviral vector for expression of FLAG-tagged EBF3 was generated in two steps. First, primer sequences encoding the FLAG tag, a Gly-Ala-Leu-Thr spacer, and a linker SpeI site were ligated into BS-KS(+) to produce 5′-GTCGACCATGGATTACAAGGACGACGACGATAAAGGTGCTCTGACTAGT-3′. EBF3 was amplified with the EBF3 cDNA clone (I.M.A.G.E clone IRCMp5012D0321D, Source Bioscience), Pfu Ultra II Fusion HS DNA polymerase (Agilent Technologies), and primers 1 and 2 (Table S4). The amplified fragment was digested with SpeI and NotI for ligations into a similarly digested BSK-FLAG vector to make BSK-FLAG-EBF3(wt). FLAG-EBF3 was excised as a SalI-NotI fragment and subcloned into the retroviral vector MSCV-IRES-MCFP (S.J.W. and J.H., manuscript in preparation), which was digested with XhoI and NotI. Mutations were introduced into EBF3 according to a protocol based on that in Fitzsimmons et al. In brief, each mutant sense or antisense primer was used together with primer 1 or 2 to amplify EBF3 from BSK-FLAG-EBF3. Fragments were gel purified, and 5′ and 3′ fragments were combined and amplified with primers 1 and 2 alone. PCR fragments were digested with BglII and BstEII for ligation into the similarly digested BSK-FLAG-EBF3. Inserts were excised with SalI and NotI for ligation into MSCV-IRES-MCFP. The T7 epitope tag was added upstream of wild-type EBF3 via subcloning of the SpeI-NotI fragment of BSK-FLAG-EBF3 into NheI-NotI-digested BSK-T73-CHD4. The SalI-NotI fragment including T7-EBF3 was subcloned into XhoI-NotI-digested MSCV-IRES-GFP2α (provided by P. Marrack). All plasmids were sequenced for confirmation of the correct cloning and mutagenesis. Culture of the μM2.21 plasmacytoma cell line, infection with the retroviruses, and detection of mIgM by flow cytometry were described previously. Whole-cell extracts were obtained via sorting of GFP⁺, mCFP⁺, or double-positive populations, washing once in cold PBS (pH 7.5), and then cell lysing for 15 min on ice in a mixture of radioimmunoprecipitation assay buffer (25 mM Tris-HCL [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 1% SDS) containing 1× HALT and protease inhibitors (Thermo Fisher) and an additional 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 5 mM NaF, 5 mM DTT, and 1 μg/mL Pepstatin A prepared fresh. Total protein concentrations were determined by Bradford assay. For mIgM expression, p values comparing column mean differences for individual constructs across their expression dosages were obtained via two-way ANOVA with Tukey’s correction for multiple comparisons. Similar analysis was performed to compare main effects across the mean of each construct. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗p < 0.05. Significance was set to p < 0.05. (A) Cells were sorted for low, medium, or high levels of GFP expression (a correlate for EBF protein level) across non-overlapping decades representing 1-, 10-, and 100-fold expression. Histograms represent three independent experiments. (B) Quantification and statistical analysis of mIgM expression on plasmacytoma cells in response to wild-type or mutant EBF3 across increasing levels of expression. Error bars represent mean values ± SD of three independent experiments.

Figure 4

EBF3 Forms Multimers with EBF3 Mutants In Vitro Western blotting (WB, top four lanes) and co-immunoprecipitation (co-IP, bottom four lanes) of EBF3 in retrovirally infected plasmacytoma cells. For western blots, 20 μg of total protein was mixed with 5% 2-mercaptoethanol and 1× Laemmli buffer and resolved on a mini-PROTEAN or Criterion 4%–20% TGX gel (Bio-Rad) at 80–120 V for 1–2 hr. Proteins were then wet transferred onto a 0.45 μm Amersham Protran nitrocellulose membrane (GE Healthcare Life Sciences) for 2.5 hr at 4°C, blocked for 2 hr in 5% milk at room temperature, and stained overnight with primary antibodies in a 5% milk solution in 1× PBS and 0.1% TWEEN. The next day, membranes were washed three times with 1× PBS and 0.1% Tween20, stained with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hr at room temperature, washed three times with 1× PBS and 0.1% TWEEN, and then washed four times with 1× PBS. Membranes were then incubated with ECL-Plus WB substrate (Thermo Scientific) for 5 min and imaged on a Typhoon FLA9500 (GE Healthcare Life Sciences). For co-IP, μM2.21 cells were infected with listed constructs plus empty-vector control or co-infected with retroviruses expressing N-terminal T7-epitope-tagged EBF3 together with N-terminal FLAG-EBF3, -(R163P), -(P177L), or -(K193N). For all conditions, double-positive GFP⁺mCFP⁺ cells were sorted and whole-cell extracts were prepared as described for WB. For each condition, 100 μg of total protein was incubated at 4°C overnight with 1 μL of antibody (anti-FLAG, Rockland Rb-600-401-383; anti-T7, Novagen 69522-3). The next day, 25 μL of Protein A/G magnetic beads (Thermo Scientific) was washed and added to each sample, incubated for 3 hr at 4°C, washed twice with lysis buffer and inhibitors, and then eluted with 1× Laemmli buffer and 5% 2-ME for 10 min at 90°C. Samples were loaded onto mini-PROTEAN gels (Bio-Rad). WB was performed as described previously. Primary antibodies used were anti-EBF1(Abnova H00001879-M01), anti-β-actin (Abcam ab8227), anti-T7-TAG HRP (Novagen 69048-3), and anti-FLAG M2 peroxidase conjugate (Sigma A8592). Secondary antibodies used were anti-mouse HRP conjugate (Promega w402b) and anti-rabbit HRP conjugate (Promega w401b). All primary antibodies were used at 1:1,000, and secondary antibodies were used at 1:10,000. WB and co-IP images were generated from the same sample and represent two independent experiments.