Project description:Although Mendelian disorders are overwhelmingly attributed to protein-coding pathogenic variants, a majority of unsolved cases do not harbor obvious causal pathogenic variants in the coding sequence, suggesting a potential non-coding etiology. However, classification of pathogenicity in non-coding sequence remains prohibitive due to a vastly increased search space and the lack of a standardized rubric for interpretation. Here, we present an integrated single cell multiomic framework to nominate pathogenic non-coding variants for the congenital cranial dysinnervation disorders (CCDDs). The CCDDs are Mendelian neurodevelopmental disorders that result from aberrant development of cranial motor neurons in the embryonic brainstem. We created a non-coding reference atlas of single cell chromatin accessibility profiles for 86,089 embryonic mouse cranial motor neurons (cMNs). We found that high-quality single cell ATAC-seq (scATAC) profiles alone were a strong predictor of enhancement (64% in vivo validation rate). To further aid in interpretation, we integrated single cell histone modification and gene expression information to distinguish individual enhancers and their cognate genes. Relatively subtle differences in cellular composition of input data often led to substantial differences in predicted enhancer strength, cognate gene, and tissue of activity. Next, we mapped candidate non-coding variants from 899 whole genome sequences from 270 CCDD pedigrees to the murine cMN-specific regulatory elements and trained a machine learning classifier to accurately predict the functional effects of patient variants within these elements. We then performed high coverage scATACseq and site-specific footprinting analysis on an allelic series of CRISPR-humanised mice to validate our machine learning predictions and render important clues to the mode of pathogenicity. Finally, we performed peak- and gene-centric allelic aggregation to nominate non-coding variants, including those regulating MN1 and EBF3, respectively. Altogether this work extends non-coding variant analysis to Mendelian disease and presents a generalizable framework for nominating novel non-coding variants in other rare disorders.

Project description:The Gabriella Miller Kids First Pediatric Research Program (<a href="https://www.commonfund.nih.gov/KidsFirst">Gabriella Miller Kids First Pediatric Research Program</a>) (Kids First) is a trans-NIH effort initiated in response to the <a href="https://www.govtrack.us/congress/bills/113/hr2019">2014 Gabriella Miller Kids First Research Act</a> and supported by the NIH Common Fund. This program focuses on gene discovery in pediatric cancers and structural birth defects and the development of the Gabriella Miller Kids First Pediatric Data Resource (Kids First Data Resource). Both, childhood cancers and structural birth defects are critical and costly conditions associated with substantial morbidity and mortality. Elucidating the underlying genetic etiology of these diseases has the potential to profoundly improve preventative measures, diagnostics, and therapeutic interventions. Whole Genome Sequence (WGS) and phenotypic data from this study are accessible through dbGaP and <a href="https://kidsfirstdrc.org">kidsfirstdrc.org</a>, where other Kids First datasets can also be accessed. Goals of this ongoing study are to identify novel "congenital cranial dysinnervation disorder" (CCDD) genes and define the role of the wildtype and mutant genes in normal and aberrant development. The umbrella term (CCDD) refers to congenital birth defects with malformation of one or more cranial nerves, typically resulting in limitations of eye and/or face movement. Examples of CCDDs include congenital fibrosis of the extraocular muscles (CFEOM), congenital ptosis, Duane retraction syndrome (DRS), horizontal gaze palsy with progressive scoliosis (HGPPS), congenital 3rd, 4th or 6th nerve palsies, Moebius syndrome (MBS), and hereditary congenital facial paresis (HCFP). In some cases, anosmia, and disorders of hearing, sucking, chewing, swallowing, and breathing may also be classified as CCDDs. CCDDs can be accompanied by additional birth defects such as intellectual and social disabilities, developmental delays, limb anomalies, and cardiac, GI, and GU disorders. The genetic basis of multiple CCDDs has been determined, and the gene mutations typically alter cranial motor neuron identity or function, or perturb axon growth and guidance. Despite these successes, the genetic etiologies of many inherited CCDDs remain unidentified.

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Noncoding Mendelian epigenomics - single cell ATACseq mouse cMN data

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