To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype–phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.

DEAF1 is a transcriptional regulator associated with autoimmune and neurological disorders and is known to bind TTCG motifs. To further ascertain preferred DEAF1 DNA ligands, we screened a random oligonucleotide library containing an “anchored” CpG motif. We identified a binding consensus that generally conformed to a repeated TTCGGG motif, with the two invariant CpG dinucleotides separated by 6–11 nucleotides. Alteration of the consensus surrounding the dual CpG dinucleotides, or cytosine methylation of a single CpG half-site, eliminated DEAF1 binding. A sequence within the Htr1a promoter that resembles the binding consensus but contains a single CpG motif was confirmed to have low affinity binding with DEAF1. A DEAF1 binding consensus was identified in the EIF4G3 promoter and ChIP assay showed endogenous DEAF1 was bound to the region. We conclude that DEAF1 preferentially binds variably spaced and unmethylated CpG-containing half-sites when they occur within an appropriate consensus.

De novo deleterious and heritable biallelic mutations in the DNA binding domain (DBD) of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of disorders termed DEAF1-associated neurodevelopmental disorders (DAND). RNA-sequencing using hippocampal RNA from mice with conditional deletion of Deaf1 in the central nervous system indicate that loss of Deaf1 activity results in the altered expression of genes involved in neuronal function, dendritic spine maintenance, development, and activity, with reduced dendritic spines in hippocampal regions. Since DEAF1 is not a dosage-sensitive gene, we assessed the dominant negative activity of previously identified de novo variants and a heritable recessive DEAF1 variant on selected DEAF1-regulated genes in 2 different cell models. While no altered gene expression was observed in cells over-expressing the recessive heritable variant, the gene expression profiles of cells over-expressing de novo variants resulted in similar gene expression changes as observed in CRISPR-Cas9-mediated DEAF1-deleted cells. Altered expression of DEAF1-regulated genes was rescued by exogenous expression of WT-DEAF1 but not by de novo variants in cells lacking endogenous DEAF1. De novo heterozygous variants within the DBD of DEAF1 were identified in 10 individuals with a phenotypic spectrum including autism spectrum disorder, developmental delays, sleep disturbance, high pain tolerance, and mild dysmorphic features. Functional assays demonstrate these variants alter DEAF1 transcriptional activity. Taken together, this study expands the clinical phenotypic spectrum of individuals with DAND, furthers our understanding of potential roles of DEAF1 on neuronal function, and demonstrates dominant negative activity of identified de novo variants.