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Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline—NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications. The detection of structural variants can be difficult with short-read sequencing technology, especially when variants are highly complex. Here, the authors use a MinION nanopore sequencer to analyse two patient genomes and develop NanoSV to map known and novel structural variants in long read data.

Copy number variants (CNVs) are major contributors to genetic diversity and disease. While standardized methods, such as the genome analysis toolkit (GATK), exist for detecting short variants, technical challenges have confounded uniform large-scale CNV analyses from whole-exome sequencing (WES) data. Given the profound impact of rare and de novo coding CNVs on genome organization and human disease, we developed GATK-gCNV, a flexible algorithm to discover rare CNVs from sequencing read-depth information, complete with open-source distribution via GATK. We benchmarked GATK-gCNV in 7,962 exomes from individuals in quartet families with matched genome sequencing and microarray data, finding up to 95% recall of rare coding CNVs at a resolution of more than two exons. We used GATK-gCNV to generate a reference catalog of rare coding CNVs in WES data from 197,306 individuals in the UK Biobank, and observed strong correlations between per-gene CNV rates and measures of mutational constraint, as well as rare CNV associations with multiple traits. In summary, GATK-gCNV is a tunable approach for sensitive and specific CNV discovery in WES data, with broad applications. GATK-gCNV uses a probabilistic model and inference framework to discover rare copy number variants (CNVs) from sequencing read-depth information. This algorithm is used to generate a reference catalog of rare coding CNVs in exome sequencing data from UK Biobank.

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF’s RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted. Ultra-deep mapping of genome organization uncovers precise nuclear compartments and diffuse CTCF loops. This work demonstrates that compartment domains segregate the 5′ and 3′ ends of genes and that CTCF loops create proximal structures.

Overexpression of all 29 human transcripts of a region of the 16p11.2 chromosome in zebrafish embryos identifies KCTD13 as the message inducing the microcephaly phenotype associated with 16p11.2 duplication, whereas its suppression yields the macrocephalic phenotype associated with the reciprocal deletion, suggesting that KCTD13 is a major driver for the neurodevelopmental phenotypes associated with the 16p11.2 copy number variants. Gene dosage in psychiatric disease Copy number variants (CNVs) make an important contribution to genetic disorders, and some CNVs have been shown to have reciprocal phenotypic effects. For instance, duplication of chromosomal region 16p11.2 has been linked to autism, schizophrenia and microcephaly, and reciprocal deletion to autism, obesity and macrocephaly. By manipulating levels of expression — in pairwise combination — of zebrafish orthologues in this genomic interval, Nicholas Katsanis and colleagues identified KCTD13 as the locus that can recapitulate the macro- and microcephalic phenotype, which they show is underpinned by a proliferative defect. Together with further human genetic data, these results suggest that KCTD13 is a major driver for the neurodevelopmental phenotypes associated with 16p11.2 duplication/deletion. The approach used here also offers a way of identifying other dosage-sensitive loci. Copy number variants (CNVs) are major contributors to genetic disorders 1 . We have dissected a region of the 16p11.2 chromosome—which encompasses 29 genes—that confers susceptibility to neurocognitive defects when deleted or duplicated 2 , 3 . Overexpression of each human transcript in zebrafish embryos identified KCTD13 as the sole message capable of inducing the microcephaly phenotype associated with the 16p11.2 duplication 2 , 3 , 4 , 5 , whereas suppression of the same locus yielded the macrocephalic phenotype associated with the 16p11.2 deletion 5 , 6 , capturing the mirror phenotypes of humans. Analyses of zebrafish and mouse embryos suggest that microcephaly is caused by decreased proliferation of neuronal progenitors with concomitant increase in apoptosis in the developing brain, whereas macrocephaly arises by increased proliferation and no changes in apoptosis. A role for KCTD13 dosage changes is consistent with autism in both a recently reported family with a reduced 16p11.2 deletion and a subject reported here with a complex 16p11.2 rearrangement involving de novo structural alteration of KCTD13 . Our data suggest that KCTD13 is a major driver for the neurodevelopmental phenotypes associated with the 16p11.2 CNV, reinforce the idea that one or a small number of transcripts within a CNV can underpin clinical phenotypes, and offer an efficient route to identifying dosage-sensitive loci.

Variably protease-sensitive prionopathy (VPSPr) is a rare, atypical subtype of prion disease currently classified as sporadic. We performed exome sequencing and targeted sequencing of PRNP non-coding regions on genomic DNA from autopsy-confirmed VPSPr patients (N = 67) in order to search for a possible genetic cause. Our search identified no potentially causal variants for VPSPr. The common polymorphism PRNP M129V was the largest genetic risk factor for VPSPr, with an odds ratio of 7.0. Other variants in and near PRNP exhibited association to VPSPr risk only in proportion to their linkage disequilibrium with M129V, and upstream expression quantitative trait loci showed no evidence of independent association to VPSPr risk. We cannot rule out the possibility of causal variants hiding in genomic regions or classes of genetic variation that our search did not canvas. Nevertheless, our data support the classification of VPSPr as a sporadic prion disease.

Reciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition. 16p11.2 CNVs are associated with neurodevelopmental disorders. Here, the authors show that 16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro.

Translocation of chromosomes can result in disruption of genes. In this case report, a sequencing approach was used to identify the cause and effect of a translocation within 13 days, a period consistent with use of the approach in prenatal diagnosis. Deep sequencing of the whole genome holds diagnostic promise but is currently thought to be impractical for routine prenatal care. In contrast, large-insert mate-pair, or jumping-library, sequencing provides a tractable approach for immediate clinical application and could complement conventional prenatal diagnostics. The risk of major structural birth defects among live births in the United States is approximately 3% 1 and is associated with inherited or de novo genetic rearrangements and mutations as well as with maternal factors, such as advanced age, certain clinical conditions, and exposure to teratogenic factors. Approximately 1 in 2000 prenatal cases analyzed with conventional karyotyping has a . . .

Age-related macular degeneration (AMD) is a prevalent cause of vision loss in the elderly with limited therapeutic options. A single chromosomal region around the complement factor H gene ( CFH ) is reported to explain nearly 25% of genetic AMD risk. Here, we used association testing, statistical finemapping and conditional analyses in 12,495 AMD cases and 461,686 controls to deconvolute four major CFH haplotypes that convey protection from AMD. We show that beyond CFH , two of these are explained by Finn-enriched frameshift and missense variants in the CFH modulator CFHR5 . We demonstrate through a FinnGen sample recall study that CFHR5 variant carriers exhibit dose-dependent reductions in serum levels of the CFHR5 gene product FHR-5 and two functionally related proteins at the locus. Genetic reduction in FHR-5 correlates with higher complement activation capacity and a thicker retinal photoreceptor layer. Our results propose therapeutic downregulation of FHR-5 as promising to prevent or treat AMD. This study in the Finnish population reveals genetic reduction of the complement factor CFHR5 as enhancing retinal health and reducing the risk for age-related macular degeneration (AMD), suggesting new strategies for AMD prevention and treatment.

Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR , LIPA , MLH1 and MAPT . Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions. Drugs that modify RNA splicing are promising treatments for many genetic diseases. Here the authors show that deep learning strategies can predict drug targets, strongly supporting the use of in silico approaches to expand the therapeutic potential of drugs that modulate RNA splicing.

Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 ( ELP1 ) gene. This mutation results in a tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9 ; Elp1 Δ20/flox . This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.