Person:

Talkowski, Michael

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.

Copy number variants (CNVs) are major contributors to genetic disorders1. We have dissected a region of the 16p11.2 chromosome—which encompasses 29 genes—that confers susceptibility to neurocognitive defects when deleted or duplicated2, 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 duplication2, 3, 4, 5, whereas suppression of the same locus yielded the macrocephalic phenotype associated with the 16p11.2 deletion5, 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.

Background: Autism spectrum disorder (ASD) is reported in 30 to 60% of patients with tuberous sclerosis complex (TSC) but shared genetic mechanisms that exist between TSC-associated ASD and idiopathic ASD have yet to be determined. Through the small G-protein Rheb, the TSC proteins, hamartin and tuberin, negatively regulate mammalian target of rapamycin complex 1 (mTORC1) signaling. It is well established that mTORC1 plays a pivotal role in neuronal translation and connectivity, so dysregulation of mTORC1 signaling could be a common feature in many ASDs. Pam, an E3 ubiquitin ligase, binds to TSC proteins and regulates mTORC1 signaling in the CNS, and the FBXO45-Pam ubiquitin ligase complex plays an essential role in neurodevelopment by regulating synapse formation and growth. Since mounting evidence has established autism as a disorder of the synapses, we tested whether rare genetic variants in TSC1, TSC2, MYCBP2, RHEB and FBXO45, genes that regulate mTORC1 signaling and/or play a role in synapse development and function, contribute to the pathogenesis of idiopathic ASD. Methods: Exons and splice junctions of TSC1, TSC2, MYCBP2, RHEB and FBXO45 were resequenced for 300 ASD trios from the Simons Simplex Collection (SSC) using a pooled PCR amplification and next-generation sequencing strategy, targeted to the discovery of deleterious coding variation. These detected, potentially functional, variants were confirmed by Sanger sequencing of the individual samples comprising the pools in which they were identified. Results: We identified a total of 23 missense variants in MYCBP2, TSC1 and TSC2. These variants exhibited a near equal distribution between the proband and parental pools, with no statistical excess in ASD cases (P > 0.05). All proband variants were inherited. No putative deleterious variants were confirmed in RHEB and FBXO45. Three intronic variants, identified as potential splice defects in MYCBP2 did not show aberrant splicing upon RNA assay. Overall, we did not find an over-representation of ASD causal variants in the genes studied to support them as contributors to autism susceptibility. Conclusions: We did not observe an enrichment of rare functional variants in TSC1 and TSC2 genes in our sample set of 300 trios.

Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. In most models, little is known concerning structural integrity of the transgene, precise site of integration, or its impact on the host genome. We previously used whole-genome and targeted sequencing approaches to reconstruct transgene structure and integration sites in models of Huntington’s disease, revealing complex structural rearrangements that can result from transgenesis. Here, we demonstrate in the R6/2 mouse, a widely used Huntington’s disease model, that integration of a rearranged transgene with coincident deletion of 5,444 bp of host genome within the gene Gm12695 has striking molecular consequences. Gm12695, the function of which is unknown, is normally expressed at negligible levels in mouse brain, but transgene integration has resulted in cortical expression of a partial fragment (exons 8–11) 3’ to the transgene integration site in R6/2. This transcript shows significant expression among the extensive network of differentially expressed genes associated with this model, including synaptic transmission, cell signalling and transcription. These data illustrate the value of sequence-level resolution of transgene insertions and transcription analysis to inform phenotypic characterization of transgenic models utilized in therapeutic research.

Background: Structural variation (SV) influences genome organization and contributes to human disease. However, the complete mutational spectrum of SV has not been routinely captured in disease association studies. Results: We sequenced 689 participants with autism spectrum disorder (ASD) and other developmental abnormalities to construct a genome-wide map of large SV. Using long-insert jumping libraries at 105X mean physical coverage and linked-read whole-genome sequencing from 10X Genomics, we document seven major SV classes at ~5 kb SV resolution. Our results encompass 11,735 distinct large SV sites, 38.1% of which are novel and 16.8% of which are balanced or complex. We characterize 16 recurrent subclasses of complex SV (cxSV), revealing that: (1) cxSV are larger and rarer than canonical SV; (2) each genome harbors 14 large cxSV on average; (3) 84.4% of large cxSVs involve inversion; and (4) most large cxSV (93.8%) have not been delineated in previous studies. Rare SVs are more likely to disrupt coding and regulatory non-coding loci, particularly when truncating constrained and disease-associated genes. We also identify multiple cases of catastrophic chromosomal rearrangements known as chromoanagenesis, including somatic chromoanasynthesis, and extreme balanced germline chromothripsis events involving up to 65 breakpoints and 60.6 Mb across four chromosomes, further defining rare categories of extreme cxSV. Conclusions: These data provide a foundational map of large SV in the morbid human genome and demonstrate a previously underappreciated abundance and diversity of cxSV that should be considered in genomic studies of human disease. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1158-6) contains supplementary material, which is available to authorized users.

Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell clones, we performed whole-genome sequencing at high coverage in order to assess the degree of mutagenesis across the entire genome. In both types of clones, we found that off-target mutations attributable to the nucleases were very rare. From this analysis, we suggest that, although some cell types may be at risk for off-target mutations, the incidence of such effects in human pluripotent stem cells may be sufficiently low and thus not a significant concern for disease modeling and other applications.

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.

SUMMARY Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals1–3. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery4,5. Despite a clear heritable component, the genetic etiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds) that segregates with MVP in the family. Morpholino knockdown of the zebrafish homolog dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 mRNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells, and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1+/− mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs as well as in Dchs1+/− mouse MVICs result in altered migration and cellular patterning, supporting these processes as etiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Recurrent, reciprocal genomic disorders resulting from non-allelic homologous recombination (NAHR) between near-identical segmental duplications (SDs) are a major cause of human disease, often producing phenotypically distinct syndromes. The genomic architecture of flanking SDs presents a significant challenge for modeling these syndromes; however, the capability to efficiently generate reciprocal copy number variants (CNVs) that mimic NAHR would represent an invaluable modeling tool. We describe here a CRISPR/Cas9 genome engineering method, Single-guide-CRISPR/Cas-targeting-Of-Repetitive-Elements (SCORE), to model reciprocal genomic disorders and demonstrate its capabilities by generating reciprocal CNVs of 16p11.2 and 15q13.3, including alteration of one copy-equivalent of the SDs that mediate NAHR in vivo. The method is reproducible and RNAseq reliably clusters transcriptional signatures from human subjects with in vivo CNV and their corresponding in vitro models. This new approach will provide broad applicability for the study of genomic disorders and, with further development, may also permit efficient correction of these defects.

The integration of electronic medical records (EMRs) and genomic research has become a major component of efforts to advance personalized and precision medicine. The Electronic Medical Records and Genomics (eMERGE) network, initiated in 2007, is an NIH-funded consortium devoted to genomic discovery and implementation research by leveraging biorepositories linked to EMRs. In its most recent phase, eMERGE III, the network is focused on facilitating implementation of genomic medicine by detecting and disclosing rare pathogenic variants in clinically relevant genes. Partners Personalized Medicine (PPM) is a center dedicated to translating personalized medicine into clinical practice within Partners HealthCare. One component of the PPM is the Partners Healthcare Biobank, a biorepository comprising broadly consented DNA samples linked to the Partners longitudinal EMR. In 2015, PPM joined the eMERGE Phase III network. Here we describe the elements of the eMERGE clinical center at PPM, including plans for genomic discovery using EMR phenotypes, evaluation of rare variant penetrance and pleiotropy, and a novel randomized trial of the impact of returning genetic results to patients and clinicians.