Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Mutational scanning of CRX classifies clinical variants and reveals biochemical properties of the transcriptional effector domain.

The transcription factor (TF) cone-rod homeobox (CRX) is essential for the differentiation and maintenance of photoreceptor cell identity. Several human CRX variants cause degenerative retinopathies, but most are variants of uncertain significance. We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitutions in CRX using a cell-based transcriptional reporter assay, curating a high-confidence list of nearly 2000 variants with altered transcriptional activity. In the structured homeodomain, activity scores closely aligned to a predicted structure and demonstrated position-specific constraints on amino acid substitution. In contrast, the intrinsically disordered transcriptional effector domain displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. These compositional constraints were consistent with the acidic exposure model of transcriptional activation. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, identifying pathogenic variants with high specificity and moderate sensitivity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate utility for integration into the clinical variant classification pipeline.

Pathogenic variants in CRX have distinct cis-regulatory effects on enhancers and silencers in photoreceptors.

Dozens of variants in the gene for the homeodomain transcription factor (TF) cone-rod homeobox (CRX) are linked with human blinding diseases that vary in their severity and age of onset. How different variants in this single TF alter its function in ways that lead to a range of phenotypes is unclear. We characterized the effects of human disease-causing variants on CRX cis-regulatory function by deploying massively parallel reporter assays (MPRAs) in mouse retina explants carrying knock-ins of two variants, one in the DNA-binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation in these mutant Crx retinas corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, and p.E168d2 has distinct effects on silencers. Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are derepressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci are partially predictive of episomal MPRA activity, and distal elements whose accessibility increases later in retinal development are enriched for CREs with silencer activity. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to misregulation of similar sets of enhancers while having a qualitatively different impact on silencers.

Mutational scanning of CRX classifies clinical variants and reveals biochemical properties of the transcriptional effector domain.

Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, CRX comprises an ordered DNA-binding homeodomain and an intrinsically disordered transcriptional effector domain. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity. We calculated a statistical confidence for each activity score derived from multiple independent measurements of each variant marked by unique sequence barcodes, curating a high-confidence list of nearly 2,000 variants with significantly altered transcriptional activity compared to wild-type CRX. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, and determined that activity scores could be used to identify pathogenic variants with high specificity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Per-position average activity scores closely aligned to a predicted structure of the ordered homeodomain and demonstrated position-specific residue requirements. The intrinsically disordered transcriptional effector domain, by contrast, displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. The observed compositional constraints of the effector domain were consistent with the acidic exposure model of transcriptional activation. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate clinical utility for variant classification.

Pathogenic variants in Crx have distinct cis-regulatory effects on enhancers and silencers in photoreceptors.

Dozens of variants in the photoreceptor-specific transcription factor (TF) CRX are linked with human blinding diseases that vary in their severity and age of onset. It is unclear how different variants in this single TF alter its function in ways that lead to a range of phenotypes. We examined the effects of human disease-causing variants on CRX cis-regulatory function by deploying massively parallel reporter assays (MPRAs) in live mouse retinas carrying knock-ins of two variants, one in the DNA binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation caused by the variants corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, while p.E168d2 has stronger effects on silencers. Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are de-repressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci were partially predictive of episomal MPRA activity, and silencers were notably enriched among distal elements whose accessibility increases later in retinal development. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to misregulation of similar sets of enhancers, while having a qualitatively different impact on silencers.

Delineation of the 1q24.3 microdeletion syndrome provides further evidence for the potential role of non-coding RNAs in regulating the skeletal phenotype.

Microdeletions within 1q24 have been associated with growth deficiency, varying intellectual disability, and skeletal abnormalities. The candidate locus responsible for the various phenotypic features of this syndrome has previously been predicted to lie in the area of 1q24.3, but molecular evidence of the causative gene remains elusive. Here, we report two additional patients carrying the smallest reported 1q24 deletion to date. Patient 1 exhibited intrauterine growth retardation, shortening of the long bones, frontal bossing, microstomia, micrognathia, and a language acquisition delay. Her mother, Patient 2, displayed a broad forehead and nasal bridge, thick supraorbital ridges, and toe brachydactyly, along with learning disability and language acquisition delay. The microdeletion encompasses a 94 Kb region containing exon 14 and portions of the surrounding introns of the gene encoding dynamin 3 (DNM3), resulting in an in-frame loss of 38 amino acids. This microdeletion site also contains a long non-coding RNA (DNM3OS) and three microRNAs (miR-214, miR-199A2, and miR-3120). Following culture of patient-derived and control fibroblasts, molecular analyses were performed to determine expression levels of genes affected by the heterozygous deletion. Results show decreased expression of DNM3OS and miR-214-3p in patient fibroblasts cultured in an osteogenic induction medium. Overall, our data provide further evidence to support a functional role for non-coding RNAs in regulating the skeletal phenotype, and the potential of a functionally-impaired DNM3 protein causing the non-skeletal disease pathogenesis.

Synthetic and genomic regulatory elements reveal aspects of cis-regulatory grammar in mouse embryonic stem cells.

In embryonic stem cells (ESCs), a core transcription factor (TF) network establishes the gene expression program necessary for pluripotency. To address how interactions between four key TFs contribute to cis-regulation in mouse ESCs, we assayed two massively parallel reporter assay (MPRA) libraries composed of binding sites for SOX2, POU5F1 (OCT4), KLF4, and ESRRB. Comparisons between synthetic cis-regulatory elements and genomic sequences with comparable binding site configurations revealed some aspects of a regulatory grammar. The expression of synthetic elements is influenced by both the number and arrangement of binding sites. This grammar plays only a small role for genomic sequences, as the relative activities of genomic sequences are best explained by the predicted occupancy of binding sites, regardless of binding site identity and positioning. Our results suggest that the effects of transcription factor binding sites (TFBS) are influenced by the order and orientation of sites, but that in the genome the overall occupancy of TFs is the primary determinant of activity.

Transcription factors are proteins that flip genetic switches; their role is to control when and where genes are active. They do this by binding to short stretches of DNA called cis-regulatory sequences. Each sequence can have several binding sites for different transcription factors, but it is largely unclear whether the transcription factors binding to the same regulatory sequence actually work together. It is possible that each transcription factor may work independently and there only needs to be critical mass of transcription factors bound to throw the genetic switch. If this is the case, the most important features of a cis-regulatory sequence should be the number of binding sites it contains, and how tightly the transcription factors bind to those sites. The more transcription factors and the more strongly they bind, the more active the gene should be. An alternative option is that certain transcription factors may work better together, enhancing each other's effects such that the total effect is more than the sum of its parts. If this is true, the order, orientation and spacing of the binding sites within a sequence should matter more than the number. One way to investigate to distinguish between these possibilities is to study mouse embryonic stem cells, which have a core set of four transcription factors. Looking directly at a real genome, however, can be confusing and it is difficult to measure the effects of different cis-regulatory sequences because genes differ in so many other ways. To tackle this problem, King et al. created a synthetic set of cis-regulatory sequences based on the four core transcription factors found in mouse stem cells. The synthetic set had every combination of two, three or four of the binding sites, with each site either facing forwards or backwards along the DNA strand. King et al. attached each of the synthetic cis-regulatory sequences to a reporter gene to find out how well each sequence performed. This revealed that the cis-regulatory sequences with the most binding sites and the tightest binding affinities work best, suggesting that transcription factors mainly work independently. There was evidence of some interaction between some transcription factors, because, of the synthetic sequences with four binding sites, some worked better than others, and there were patterns in the most effective binding site combinations. However, these effects were small and when King et al. went on to test sequences from the real mouse genome, the most important factor by far was the number of binding sites. Synthetic libraries of DNA sequences allow researchers to examine gene regulation more clearly than is possible in real genomes. Yet this approach does have its limitations and it is impossible to capture every type of cis-regulatory sequence in one library. The next step to extend this work is to combine the two approaches, taking sequences from the real genome and manipulating them one by one. This could help to unravel the rules that govern how cis-regulatory sequences work in real cells.