Elizabeth C. Engle

INTRODUCTION: Despite major advances in genomic sequencing and analytical pipelines for rare disorders, more than 50% of affected individuals remain without a molecular diagnosis. One challenge is the interpretation of inherited variants exhibiting incomplete penetrance (IP). MATERIALS AND METHODS: We developed a pipeline to systematically investigate inherited variants as the cause of rare disease in 4,325 families, consisting of affected probands and unaffected parents, from the Broad Center for Mendelian Genomics, part of the GREGoR Consortium. As a proof-of-concept of further omics follow-up, epigenomic and transcriptomic analyses were performed in one family harboring an inherited disease-causing frameshift variant in CHD8 . RESULTS: We identified 81 families where the affected proband harbored a putative disease-causing variant inherited from a reportedly unaffected parent. Clinical re-evaluation identified 30 families in which an inherited variant was considered diagnostic, including six previously missed diagnoses. In this cohort, inherited IP variants account for 1.6% of the diagnosed cases, with an overall diagnostic yield of 0.4% in the cohort. Methylation analysis of the CHD8 family confirmed a positive CHD8 clinical episignature in the male proband, but also in the reportedly unaffected mother. Transcriptomic analysis showed that despite similar CHD8 haploinsufficiency, the proband and mother displayed divergent downstream transcriptional effects. DISCUSSION: This study highlights the importance of considering parentally inherited IP variants in rare disease, with at least 1 in 60 diagnostic variants inherited from a reportedly unaffected parent, and demonstrates the potential of integrating omics evaluation to improve variant interpretation.

Strabismus is a common pediatric eye misalignment and has complex genetic and environmental causes. Previous genome-wide association studies (GWAS) encountered difficulties in identifying strabismus risk variants due to heterogeneity and small samples. We performed large meta-analyses of 11 European-ancestry GWAS (7 sources), analysing broad strabismus (20,464 cases, 954,921 controls) and subtypes (esotropia/exotropia). We discovered 4 loci (e.g., NPLOC4-TSPAN10-PDE6G-FAAP100, COL6A1) for strabismus and 5 additional loci (e.g., CHRNA4, MAD1L1) for strabismus subtypes and we successfully replicated the previously reported strabismus variant near NPLOC4-TSPAN10-PDE6G-FAAP100. Using mendelian randomisation, we found genetic evidence supporting maternal smoking as a causal risk factor for strabismus in offspring.

Objective: Congenital facial weakness (CFW) disorders are a heterogeneous group of rare conditions, that present at birth, with reduced facial movement, and mask-like facies. This study utilized a multimodality approach to examine the craniofacial and intraoral phenotypes among CFW disorders: Moebius syndrome (MBS), Hereditary Congenital Facial Palsy (HCFP), β-tubulin isotype 3 syndrome (CFEOM3A-TUBB3), Carey-Fineman-Ziter syndrome (CFZS), and a group of rarer disorders (Other). Design: Prospective cohort study. Setting: Dental clinic. Participants: Sixty individuals (sex ratio 1:1, mean age 26.2 ± 17.5 years) with a diagnosis of CFW. Interventions: Deep clinical craniofacial and dental phenotyping, three-dimensional facial surface and cone-beam computed tomography scans, and cephalometric and geometric morphometric analyses. Results: CFEOM3A-TUBB3, MBS, and CFZS groups had the highest prevalence of craniofacial anomalies; HCFP individuals were least affected. CFEOM3A-TUBB3 had a higher prevalence of short lower face (75%), poor oral hygiene (100%)/decay (75%), and Class II malocclusion (87.5%). Moebius syndrome was associated with lagophthalmos (90.9%), tongue fissures (72.4%), tight/small oral orifice (51.7%), and tongue fasciculations (50%). Carey-Fineman-Ziter syndrome had oblong facial shape (100%), downward lip commissures (100%), and abnormal hearing (60%). Moderate-severe decay/gingivitis correlated with restricted oral orifice, common among patients with facial animation/sling surgery. Morphologically, the CFW cohort had a relatively small craniofacial centroid size, anthropometric measurements, and distinct craniofacial shapes for each subtype. Conclusions: Congenital facial weakness can result in abnormal craniofacial development in addition to the loss of facial movement. Multimodality phenotypic characterization of CFW disorders elucidated key clinical findings and distinct craniofacial shape segregation among the different groups.

To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously reported the identification of variants in many candidate genes. Here, we describe a parallel study that prioritized a subset of these genes (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for 17. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays. We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba. Approximately 70% to 90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)). This study nominates three strong novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 – as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

Purpose: To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously identified variants in 80 strong candidate genes. Here, we further prioritized a subset of these (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for seventeen. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays. Results: We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba . 70-90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes ( SEMA3F , OLIG2, and FRMD4B ) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)). Conclusions: This study nominates three strong novel candidate oCCDD genes ( SEMA3F , OLIG2, and FRMD4B ) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.