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The emergence of novel sequencing technologies has greatly improved the identification of structural variation, revealing that a human genome harbors tens of thousands of structural variants (SVs). Since these SVs primarily impact noncoding DNA sequences, the next challenge is one of interpretation, not least to improve our understanding of human disease etiology. However, this task is severely complicated by the intricacy of the gene regulatory landscapes embedded within these noncoding regions, their incomplete annotation, as well as their dependence on the three-dimensional (3D) conformation of the genome. Also in the context of neurodevelopmental disorders (NDDs), reports of putatively causal, noncoding SVs are accumulating and understanding their impact on transcriptional regulation is presenting itself as the next step toward improved genetic diagnosis.

Craniofacial abnormalities account for approximately one third of birth defects. The regulatory programs that build the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Clusters of coactivated enhancers and their target genes, known as superenhancers, are important in determining cell identity but have been largely unexplored in development. In this study we identified superenhancer regions unique to human embryonic craniofacial tissue. To demonstrate the importance of such regions in craniofacial development and disease, we focused on an ~600 kb noncoding region located between NPVF and NFE2L3 . We identified long range interactions with this region in both human and mouse embryonic craniofacial tissue with the anterior portion of the HOXA gene cluster. Mice lacking this superenhancer exhibit perinatal lethality, and present with highly penetrant skull defects and orofacial clefts phenocopying Hoxa2-/- mice. Moreover, we identified two cases of de novo copy number changes of the superenhancer in humans both with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and copy number changes are pathogenic in human patients. The authors discovered cluster of regulatory sequences that controls HOXA over large genomic distances and across topological domains. This cluster is required for normal craniofacial development in both humans and mice.

Recently, exome sequencing led to the identification of causal mutations in 16–31% of patients with intellectual disability (ID), leaving the underlying cause for many patients unidentified. In this context, the noncoding part of the human genome remains largely unexplored. For many long non-coding RNAs (lncRNAs) a crucial role in neurodevelopment and hence the human brain is anticipated. Here we aimed at identifying lncRNAs associated with neuronal development and ID. Therefore, we applied an integrated genomics approach, harnessing several public epigenetic datasets. We found that the presence of neuron-specific H3K4me3 confers the highest specificity for genes involved in neurodevelopment and ID. Based on the presence of this feature and GWAS hits for CNS disorders, we identified 53 candidate lncRNA genes. Extensive expression profiling on human brain samples and other tissues, followed by Gene Set Enrichment Analysis indicates that at least 24 of these lncRNAs are indeed implicated in processes such as synaptic transmission, nervous system development and neurogenesis. The bidirectional or antisense overlapping orientation relative to multiple coding genes involved in neuronal processes supports these results. In conclusion, we identified several lncRNA genes putatively involved in neurodevelopment and CNS disorders, providing a resource for functional studies.

Background Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis -regulatory elements (CREs) in 3D nuclear space. Results To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4 , which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis -regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4 . Conclusions Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases. Graphical Abstract

Structural genomic variations play an important role in human disease and phenotypic diversity. With the rise of high-throughput sequencing tools, mate-pair/paired-end/single-read sequencing has become an important technique for the detection and exploration of structural variation. Several analysis tools exist to handle different parts and aspects of such sequencing based structural variation analyses pipelines. A comprehensive analysis platform to handle all steps, from processing the sequencing data, to the discovery and visualization of structural variants, is missing. The ViVar platform is built to handle the discovery of structural variants, from Depth Of Coverage analysis, aberrant read pair clustering to split read analysis. ViVar provides you with powerful visualization options, enables easy reporting of results and better usability and data management. The platform facilitates the processing, analysis and visualization, of structural variation based on massive parallel sequencing data, enabling the rapid identification of disease loci or genes. ViVar allows you to scale your analysis with your work load over multiple (cloud) servers, has user access control to keep your data safe and is easy expandable as analysis techniques advance. URL: https://www.cmgg.be/vivar/

Background The first subjects with deficiency of mitochondrial tryptophanyl-tRNA synthetase (WARS2) were reported in 2017. Their clinical characteristics can be subdivided into three phenotypes (neonatal phenotype, severe infantile onset phenotype, Parkinson-like phenotype). Results Here, we report on a subject who presented with early developmental delay, motor weakness and intellectual disability and who was considered during several years as having a non-progressive encephalopathy. At the age of six years, she had an epileptic seizure which was treated with sodium valproate. In the months after treatment was started, she developed acute liver failure and severe progressive encephalopathy. Although valproate was discontinued, she died six months later. Spectrophotometric analysis of the oxidative phosphorylation complexes in liver revealed a deficient activity of complex III and low normal activities of the complexes I and IV. Activity staining in the BN-PAGE gel confirmed the low activities of complex I, III and IV and, in addition, showed the presence of a subcomplex of complex V. Histochemically, a mosaic pattern was seen in hepatocytes after cytochrome c oxidase staining. Using Whole Exome Sequencing two known pathogenic variants were detected in WARS2 (c.797delC, p.Pro266ArgfsTer10/ c.938 A > T, p.Lys313Met). Conclusion This is the first report of severe hepatopathy in a subject with WARS2 deficiency. The hepatopathy occurred soon after start of sodium valproate treatment. In the literature, valproate-induced hepatotoxicity was reported in the subjects with pathogenic mutations in POLG and TWNK . This case report illustrates that the course of the disease in the subjects with a mitochondrial defect can be non-progressive during several years. The subject reported here was first diagnosed as having cerebral palsy. Only after a mitochondriotoxic medication was started, the disease became progressive, and the diagnosis of a mitochondrial defect was made.

RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection of transcripts subject to nonsense-mediated decay. While broadly applicable, this protocol is particularly suited for neurodevelopmental disorders, as up to 80% of the genes in our intellectual disability and epilepsy gene panel are expressed in PBMCs. Applied to 46 affected individuals and 15 parents, RNA-seq revealed splicing defects in six of nine individuals with splice variants, allowing reclassification of seven variants. Targeted cDNA analysis confirmed aberrant splicing in four individuals but missed intron retention in two. Global analyses (FRASER, OUTRIDER, and monoallelic expression) supported findings but did not yield new diagnoses. We propose a flowchart integrating RNA-seq into diagnostic workflows. Overall, our protocol is easily implementable, captures complex splicing events, and enhances variant classification.

Chromosomal rearrangements involving the EVI1 proto-oncogene are a recurrent finding in myeloid leukemias and are indicative of a poor prognosis. Rearrangements of the EVI1 locus are often associated with monosomy 7 or cytogenetic detectable deletions of part of 7q. As EVI1 overexpression alone is not sufficient to induce leukemia, loss of a 7q tumour suppressor gene might be a required cooperating event. To test this hypothesis, we performed high-resolution array comparative genomic hybridization analysis of twelve EVI1 overexpressing patients and three EVI1 deregulated cell lines to search for 7q submicroscopic deletions. This analysis lead to the delineation of two critical regions, one of 0.39 Mb on 7q35 containing the CNTNAP2 gene and one of 1.33 Mb on chromosome bands 7q35-q36 comprising nine genes in EVI1 deregulated cell lines. These findings open the way to further studies aimed at identifying the culprit EVI1 implicated tumour suppressor genes on 7q.

Martin Zenker, Corinne Antignac, Friedhelm Hildebrandt and colleagues report that mutations in OSGEP , TP53RK , TPRKB and LAGE3 , genes encoding KEOPS-complex subunits, cause Galloway–Mowat syndrome, a recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Functional studies suggest that the phenotypes result from impaired protein translation, thus leading to endoplasmic reticulum stress and apoptosis. Galloway–Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP , TP53RK , TPRKB , and LAGE3 , genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR–Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP , TP53RK , or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.