Novel de novo variant in EBF3 is likely to impact DNA binding in a patient with a neurodevelopmental disorder and expanded phenotypes: patient report, in silico functional assessment, and review of published cases

Abstract

Pathogenic variants in EBF3 were recently described in three back-to-back publications in association with a novel neurodevelopmental disorder characterized by intellectual disability, speech delay, ataxia, and facial dysmorphisms. In this report, we describe an additional patient carrying a de novo missense variant in EBF3 (c.487C>T, p.(Arg163Trp)) that falls within a conserved residue in the zinc knuckle motif of the DNA binding domain. Without a solved structure of the DNA binding domain, we generated a homology-based atomic model and performed molecular dynamics simulations for EBF3, which predicted decreased DNA affinity for p.(Arg163Trp) compared with wild-type protein and control variants. These data are in agreement with previous experimental studies of EBF1 showing the paralogous residue is essential for DNA binding. The conservation and experimental evidence existing for EBF1 and in silico modeling and dynamics simulations to validate comparable behavior of multiple variants in EBF3 demonstrates strong support for the pathogenicity of p.(Arg163Trp). We show that our patient presents with phenotypes consistent with previously reported patients harboring EBF3 variants and expands the phenotypic spectrum of this newly identified disorder with the additional feature of a bicornuate uterus.

Keywords: bicornuate uterus; congenital strabismus; downturned corners of mouth; generalized neonatal hypotonia; hydronephrosis; hydroureter; low posterior hairline; microretrognathia; moderate global developmental delay; neurogenic bladder; poor speech; recurrent urinary tract infections; short stature; urethral stricture; vesicoureteral reflux.

Figure 1.

Patient photographs showing front and side views. The proband was noted to have mild dysmorphic features including bilateral esotropia, retrognathia, downturned corners of the mouth, and a low posterior hairline.

Figure 2.

Schematic diagram of EBF3 (NP_001005463.1) protein structure. Numbering corresponds to amino acids.

Figure 3.

p.Arg163Trp induces dynamic changes throughout EBF3 that are comparable to the validated pathogenic p.Arg163Ala and distinct from control simulations. (A) We calculated the average root-mean-square deviation (RMSD) across replicates for each simulation condition (indicated by color). Proteins with alterations at residue 163 demonstrated increased mobility throughout both monomers (indicated by light and dark gray rectangles along the abscissa) with the greatest differences among the DNA-interacting regions (black rectangles) around p.Arg163. (B) To quantify the differences between conformations, we measured the distance from residue 163 (violet sphere) to nearby phosphate atoms in the DNA backbone (orange spheres). (C) We show a comparison using one of these reference distances (larger sphere in B), demonstrating that both the wild-type (wt) and p.Lys239Ala retain stable DNA interactions, whereas both p.Arg163Ala and p.Arg163Trp lose contact with DNA. Additional distance measures are presented in Supplemental Figure S2.