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Rationale: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD) phenotype. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Objective: We used whole exome sequencing (WES) to assess the prevalence of unique, deleterious variants in the largest cohort of non-syndromic TOF patients reported to date. Methods and Results: 829 TOF patients underwent WES. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database (gnomAD) and a scaled combined annotation-dependent depletion (CADD) score of ≥20. The clustering of variants in two genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5x10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbour unique, deleterious variants. 31 changes were observed in 37 probands (4.5%; 95% confidence interval [CI]:3.2-6.1%) and included seven loss-of-function variants 22 missense variants and two in-frame indels. Sanger-sequencing of the unaffected parents of seven cases identified five de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y and p.N1875S) were subjected to functional evaluation and two showed a reduction in Jagged1-induced NOTCH signalling. FLT4 variants were found in 2.4% (95% CI:1.6-3.8%) of TOF patients, with 21 patients harbouring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy Disease. In addition to NOTCH1, FLT4 and the well-established TOF gene, TBX1, we identified potential association with variants in several other candidates including RYR1, ZFPM1, CAMTA2, DLX6 and PCM1. Conclusions: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.

Background With the decreasing cost of sequencing and the rapid developments in genomics technologies and protocols, the need for validated bioinformatics software that enables efficient large-scale data processing is growing. Findings Here we present GenPipes, a flexible Python-based framework that facilitates the development and deployment of multi-step workflows optimized for high-performance computing clusters and the cloud. GenPipes already implements 12 validated and scalable pipelines for various genomics applications, including RNA sequencing, chromatin immunoprecipitation sequencing, DNA sequencing, methylation sequencing, Hi-C, capture Hi-C, metagenomics, and Pacific Biosciences long-read assembly. The software is available under a GPLv3 open source license and is continuously updated to follow recent advances in genomics and bioinformatics. The framework has already been configured on several servers, and a Docker image is also available to facilitate additional installations. Conclusions GenPipes offers genomics researchers a simple method to analyze different types of data, customizable to their needs and resources, as well as the flexibility to create their own workflows.

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Glycine 34 to tryptophan (G34W) substitutions in H3.3 arise in ~90% of giant cell tumour of bone (GCT). Here, we show H3.3G34W is necessary for tumour formation. By profiling the epigenome, transcriptome and secreted proteome of patient samples and tumour-derived cells CRISPR/Cas9-edited for H3.3G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Singlecell transcriptomics reveals that H3.3G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population towards an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors which promotes neoplastic growth, pathological recruitment of giant osteoclasts, and bone destruction. STATEMENT OF SIGNFICANCE This study shows that H3.3G34W drives GCT tumourigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumour microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT. Research.

SummaryThe precisionFDA Truth Challenge V2 aimed to assess the state-of-the-art of variant calling in difficult-to-map regions and the Major Histocompatibility Complex (MHC). Starting with FASTQ files, 20 challenge participants applied their variant calling pipelines and submitted 64 variant callsets for one or more sequencing technologies (~35X Illumina, ~35X PacBio HiFi, and ~50X Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with the new GIAB benchmark sets and genome stratifications. Challenge submissions included a number of innovative methods for all three technologies, with graph-based and machine-learning methods scoring best for short-read and long-read datasets, respectively. New methods out-performed the 2016 Truth Challenge winners, and new machine-learning approaches combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

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The primary cilium is an outward projecting antenna-like organelle with an important role in bone mechanotransduction. The capacity to sense mechanical stimuli can affect important cellular and molecular aspects of bone tissue. Idiopathic scoliosis (IS) is a complex pediatric disease of unknown cause, defined by abnormal spinal curvatures. We demonstrate significant elongation of primary cilia in IS patient bone cells. In response to mechanical stimulation, these IS cells differentially express osteogenic factors, mechanosensitive genes, and signaling genes. Considering that numerous ciliary genes are associated with a scoliosis phenotype, among ciliopathies and knockout animal models, we expected IS patients to have an accumulation of rare variants in ciliary genes. Instead, our SKAT-O analysis of whole exomes showed an enrichment among IS patients for rare variants in genes with a role in cellular mechanotransduction. Our data indicates defective cilia in IS bone cells, which may be linked to heterogeneous gene variants pertaining to cellular mechanotransduction.