Harrison Brand

Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

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.

CHD8 Regulates Neurodevelopmental Pathways Associated with Autism Spectrum Disorder in Neural Progenitors

Significance Truncating mutation of chromodomain helicase DNA-binding protein 8 (CHD8) represents one of the strongest known risk factors for autism spectrum disorder (ASD). We mimicked the effects of such heterozygous loss-of-function mutations in neural progenitor cells and integrated RNA sequencing with genome-wide delineation of CHD8 binding. Our results reveal that the molecular mechanism by which CHD8 alters neurodevelopmental pathways may involve both direct and indirect effects, the latter involving down-regulation following CHD8 suppression. We also find that chd8 suppression in zebrafish results in macrocephaly, consistent with observations in patients harboring loss-of-function mutations. We show that reduced expression of CHD8 impacts a variety of other functionally distinct ASD-associated genes, suggesting that the diverse functions of ASD risk factors may constitute multiple means of triggering a smaller number of final common pathways. Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10−8) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10−10). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.

Loss of δ-catenin function in severe autism

Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology 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 homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA 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 aetiological 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.

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

Genomic and Functional Overlap between Somatic and Germline Chromosomal Rearrangements

Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted.

An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.This study presents a framework to evaluate rare and de novo variation from whole-genome sequencing (WGS). The work suggests that robust results from WGS studies will require large cohorts and strategies that consider the substantial multiple-testing burden.

Limited contribution of rare, noncoding variation to autism spectrum disorder from sequencing of 2,076 genomes in quartet families

Genomic studies to date in autism spectrum disorder (ASD) have largely focused on newly arising mutations that disrupt protein coding sequence and strongly influence risk. We evaluate the contribution of noncoding regulatory variation across the size and frequency spectrum through whole genome sequencing of 519 ASD cases, their unaffected sibling controls, and parents. Cases carry a small excess of de novo (1.02-fold) noncoding variants, which is not significant after correcting for paternal age. Assessing 51,801 regulatory classes, no category is significantly associated with ASD after correction for multiple testing. The strongest signals are observed in coding regions, including structural variation not detected by previous technologies and missense variation. While rare noncoding variation likely contributes to risk in neurodevelopmental disorders, no category of variation has impact equivalent to loss-of-function mutations. Average effect sizes are likely to be smaller than that for coding variation, requiring substantially larger samples to quantify this risk.

CNView: a visualization and annotation tool for copy number variation from whole-genome sequencing

Summary Copy number variation (CNV) is a major component of structural differences between individual genomes. The recent emergence of population-scale whole-genome sequencing (WGS) datasets has enabled genome-wide CNV delineation. However, molecular validation at this scale is impractical, so visualization is an invaluable preliminary screening approach when evaluating CNVs. Standardized tools for visualization of CNVs in large WGS datasets are therefore in wide demand. Methods & Results To address this demand, we developed a software tool, CNView, for normalized visualization, statistical scoring, and annotation of CNVs from population-scale WGS datasets. CNView surmounts challenges of sequencing depth variability between individual libraries by locally adapting to cohort-wide variance in sequencing uniformity at any locus. Importantly, CNView is broadly extensible to any reference genome assembly and most current WGS data types. Availability and Implementation CNView is written in R, is supported on OS X, MS Windows, and Linux, and is freely distributed under the MIT license. Source code and documentation are available from https://github.com/RCollins13/CNView Contact talkowski@chgr.mgh.harvard.edu

Evidence for secondary-variant genetic burden and non-random distribution across biological modules in a recessive ciliopathy

The influence of genetic background on driver mutations is well established; however, the mechanisms by which the background interacts with Mendelian loci remains unclear. We performed a systematic secondary-variant burden analysis of two independent Bardet-Biedl syndrome (BBS) cohorts with known recessive biallelic pathogenic mutations in one of 17 BBS genes for each individual. We observed a significant enrichment of trans-acting rare nonsynonymous secondary variants compared to either population controls or to a cohort of individuals with a non-BBS diagnosis and recessive variants in the same gene set. Strikingly, we found a significant over-representation of secondary alleles in chaperonin-encoding genes, a finding corroborated by the observation of epistatic interactions involving this complex in vivo. These data indicate a complex genetic architecture for BBS that informs the biological properties of disease modules and presents a model paradigm for secondary-variant burden analysis in recessive disorders.