Expanding the genetics and phenotypes of ocular congenital cranial dysinnervation disorders.

Purpose: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. Results: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. Conclusion: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.

Cancer immunosurveillance by gut T cells.

T cells primed in the gut influence immune responses to extraintestinal tumors.

Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis.

Hereditary congenital facial paresis type 1 (HCFP1) is an autosomal dominant disorder of absent or limited facial movement that maps to chromosome 3q21-q22 and is hypothesized to result from facial branchial motor neuron (FBMN) maldevelopment. In the present study, we report that HCFP1 results from heterozygous duplications within a neuron-specific GATA2 regulatory region that includes two enhancers and one silencer, and from noncoding single-nucleotide variants (SNVs) within the silencer. Some SNVs impair binding of NR2F1 to the silencer in vitro and in vivo and attenuate in vivo enhancer reporter expression in FBMNs. Gata2 and its effector Gata3 are essential for inner-ear efferent neuron (IEE) but not FBMN development. A humanized HCFP1 mouse model extends Gata2 expression, favors the formation of IEEs over FBMNs and is rescued by conditional loss of Gata3. These findings highlight the importance of temporal gene regulation in development and of noncoding variation in rare mendelian disease.

Annotating genetic variants to target genes using H-MAGMA.

An outstanding goal in modern genomics is to systematically predict the functional outcome of noncoding variation associated with complex traits. To address this, we developed Hi-C-coupled multi-marker analysis of genomic annotation (H-MAGMA), which builds on traditional MAGMA-a gene-based analysis tool that assigns variants to their target genes-by incorporating 3D chromatin configuration in assigning variants to their putative target genes. Applying this approach, we identified key biological pathways implicated in a wide range of brain disorders and showed its utility in complementing other functional genomic resources such as expression quantitative trait loci-based variant annotation. Here, we provide a detailed protocol for generating the H-MAGMA variant-gene annotation file by using chromatin interaction data from the adult human brain. In addition, we provide an example of how H-MAGMA is run by using genome-wide association study summary statistics of Parkinson's disease. Lastly, we generated variant-gene annotation files for 28 tissues and cell types, with the hope of contributing a resource for researchers studying a broad set of complex genetic disorders. H-MAGMA can be performed in <2 h for any cell type in which Hi-C data are available.

A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders.

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 generated 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. Seventy-five percent of elements (44 of 59) validated in an in vivo transgenic reporter assay, demonstrating that single cell accessibility is a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieved significant reduction in our variant search space and nominated candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as new candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work provides novel 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.

Advances in profiling chromatin architecture shed light on the regulatory dynamics underlying brain disorders.

Understanding the exquisitely complex nature of the three-dimensional organization of the genome and how it affects gene regulation remains a central question in biology. Recent advances in sequencing- and imaging-based approaches in decoding the three-dimensional chromatin landscape have enabled a systematic characterization of gene regulatory architecture. In this review, we outline how chromatin architecture provides a reference atlas to predict the functional consequences of non-coding variants associated with human traits and disease. High-throughput perturbation assays such as massively parallel reporter assays (MPRA) and CRISPR-based genome engineering in combination with a reference atlas opened an avenue for going beyond observational studies to experimentally validating the regulatory principles of the genome. We conclude by providing a suggested path forward by calling attention to barriers that can be addressed for a more complete understanding of the regulatory landscape of the human brain.

Novel variants in TUBA1A cause congenital fibrosis of the extraocular muscles with or without malformations of cortical brain development.

Variants in multiple tubulin genes have been implicated in neurodevelopmental disorders, including malformations of cortical development (MCD) and congenital fibrosis of the extraocular muscles (CFEOM). Distinct missense variants in the beta-tubulin encoding genes TUBB3 and TUBB2B cause MCD, CFEOM, or both, suggesting substitution-specific mechanisms. Variants in the alpha tubulin-encoding gene TUBA1A have been associated with MCD, but not with CFEOM. Using exome sequencing (ES) and genome sequencing (GS), we identified 3 unrelated probands with CFEOM who harbored novel heterozygous TUBA1A missense variants c.1216C>G, p.(His406Asp); c.467G>A, p.(Arg156His); and c.1193T>G, p.(Met398Arg). MRI revealed small oculomotor-innervated muscles and asymmetrical caudate heads and lateral ventricles with or without corpus callosal thinning. Two of the three probands had MCD. Mutated amino acid residues localize either to the longitudinal interface at which α and ß tubulins heterodimerize (Met398, His406) or to the lateral interface at which tubulin protofilaments interact (Arg156), and His406 interacts with the motor domain of kinesin-1. This series of individuals supports TUBA1A variants as a cause of CFEOM and expands our knowledge of tubulinopathies.

Recurrent Rare Copy Number Variants Increase Risk for Esotropia.

Purpose: To determine whether rare copy number variants (CNVs) increase risk for comitant esotropia. Methods: CNVs were identified in 1614 Caucasian individuals with comitant esotropia and 3922 Caucasian controls from Illumina SNP genotyping using two Hidden Markov model (HMM) algorithms, PennCNV and QuantiSNP, which call CNVs based on logR ratio and B allele frequency. Deletions and duplications greater than 10 kb were included. Common CNVs were excluded. Association testing was performed with 1 million permutations in PLINK. Significant CNVs were confirmed with digital droplet polymerase chain reaction (ddPCR). Whole genome sequencing was performed to determine insertion location and breakpoints. Results: Esotropia patients have similar rates and proportions of CNVs compared with controls but greater total length and average size of both deletions and duplications. Three recurrent rare duplications significantly (P = 1 × 10-6) increase the risk of esotropia: chromosome 2p11.2 (hg19, 2:87428677-87965359), spanning one long noncoding RNA (lncRNA) and two microRNAs (OR 14.16; 95% confidence interval [CI] 5.4-38.1); chromosome 4p15.2 (hg19, 4:25554332-25577184), spanning one lncRNA (OR 11.1; 95% CI 4.6-25.2); chromosome 10q11.22 (hg19, 10:47049547-47703870) spanning seven protein-coding genes, one lncRNA, and four pseudogenes (OR 8.96; 95% CI 5.4-14.9). Overall, 114 cases (7%) and only 28 controls (0.7%) had one of the three rare duplications. No case nor control had more than one of these three duplications. Conclusions: Rare CNVs are a source of genetic variation that contribute to the genetic risk for comitant esotropia, which is likely polygenic. Future research into the functional consequences of these recurrent duplications may shed light on the pathophysiology of esotropia.