Athurva Gore

SOMATIC CODING MUTATIONS IN HUMAN INDUCED PLURIPOTENT STEM CELLS

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.

Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming

Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.

Whole-Genome Sequencing in Autism Identifies Hot Spots for De Novo Germline Mutation

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

Whole-Genome Sequencing Analysis Reveals High Specificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs

Human iPSCs provide renewable cell sources for human biology and disease research and the potential for developing gene and cell therapy. Realization of this potential will rely in part on our ability to precisely edit or engineer the human genome in an efficient way. Recent developments in designer endonuclease technologies such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease have provided ways to significantly improve genome editing efficiency in human iPSCs. These endonucleases make a double-stranded break (DSB) at a predetermined DNA sequence and trigger natural DNA repair processes such as nonhomologous end joining (NHEJ) or homologous recombination (HR) with a donor DNA template. Among these existing approaches, RNA-guided CRISPR/Cas9 is the most user-friendly and versatile system, and it has been applied in both animal models and cell lines (Cong et al., 2013; Hsu et al., 2014; Mali et al., 2013). The most commonly used system consists of a single polypeptide endonuclease Cas9 complexed with a single guide RNA (gRNA) that provides complementarity to 20-nucleotide target DNA sequence. However, the specificity and efficiency of this approach in human iPSCs have not been studied in detail (Cong et al., 2013; Ding et al., 2013; Mali et al., 2013; Yang et al., 2013). Some analyses using cancer cell lines reported higher-than-expected levels of off-target mutagenesis by Cas9-gRNAs (Fu et al., 2013; Hsu et al., 2013), raising concerns about the practical applicability of this approach in therapeutic contexts. Some recent studies, including one on human adult stem cells, showed a minimal level of off-target effects by CRISPR/Cas9 (Schwank et al., 2013). However, these existing analyses of off-target effects and mutational load in gene-corrected stem cells have been restricted to checking predicted off target sites and are therefore limited in scope. To assess the value of this type of gene editing approach for therapeutic applications, it is critical to rigorously examine whether it is possible to generate gene-edited cell lines with minimal mutational load. To this end, we have conducted whole-genome sequencing of four iPSC clones successfully targeted at the AAVS1 locus, a “safe harbor” in the human genome that is used for stable transgene expression in a variety of contexts. To generate the lines, we used an integration-free human iPSC line, BC1, whose genomic integrity has been characterized in detail by next-generation sequencing (Cheng et al., 2012) and targeted a GFP expression cassette into the AAVS1 site with either a previously reported Cas9-gRNA combination or a pair of improved heterodimeric TALENs (Mali et al., 2013; Yan et al., 2013) (Table S1 and Supplemental Experimental Procedures available online). Twenty days after transfection of the donor plasmid and either the TALENs or Cas9-gRNA into BC1, we harvested four clones with confirmed targeted integration (hCas9-C4, hCas9-C16, TALEN-C3, and TALEN-C6; Table S1 and Supplemental Experimental Procedures) and the parental BC1 iPSCs for whole-genome sequencing. The sequencing reads, ranging from 83 Gbps to 100 Gbps from each targeted clone, were first aligned to the human hg19 reference genome to enable identification of single-nucleotide variants (SNVs) and small indels (Table S1). Our analysis identified ≥4.2 million SNVs and ≥500,000 indels in each genome (Table S1) in comparison to the hg19 reference genome, suggesting that it is a rigorous data set that covers the genome in sufficient depth to detect sequence variants. The “germ-line” variants (present in BC1 parental iPSCs and different from hg19) were readily detectable in each targeted cell line (80%%–88%), indicating that the sensitivity of variant detection in our analysis is high (Table S1). The variations from each targeted clone were then compared to the BC1 parental iPSCs to enable the generation of a list of potential variations arising during the gene editing process, which we then confirmed using genomic PCR and Sanger sequencing. We confirmed 62 out of 69 SNVs tested for an overall confirmation rate of 90%, and based on that we estimate that the total SNVs in the four iPSC clones range between 217 and 281 and that the total indels range between 7 and 12 (Table S1). Overall the genomic variation levels in TALEN- and Cas9-targeted groups were comparable. One important consideration is how many of these detected SNVs and indels were the results of off-target mutagenesis by the engineered endonucleases. To address this question, we generated a list of 3,665 (Cas9) and 238 (TALEN) putative off-target positions by using the EMBOSS fuzznuc software package. Each candidate SNV and indel was compared to this list and none of them are within a potential off-target region (Table S1), consistent with previous analyses looking at predicted off-target sites. Our analysis also shows that each SNV and indel is unique and that none of them occurred in more than one cell line. The absence of recurring mutations and the fact that none of the mutations resides in any putative off-target site by bioinformatic prediction strongly suggest that these mutations were randomly accumulated during regular cell expansion and are not direct results of off-target activities by Cas9 or TALENs. Our results from whole-genome sequencing analysis of Cas9- and TALEN-targeted human iPSC clones demonstrate that these engineered endonucleases provide efficient genome-editing tools with high specificity. It remains to be clarified whether the higher off-target rates observed in cancer cell lines are due to the overexpression of gRNAs and Cas9 protein and/or due to exacerbated and faulty DNA repair in these cell types. The higher specificity observed in human iPSCs, combined with the rapid development of next-generation sequencing technology, makes it possible to characterize and isolate high quality genome-edited stem cell clones with minimal mutational load. The guiding principle established with human iPSCs will likely be applicable to other types of stem cells and come with improvements in gene transfer and targeting efficiencies. Our current study of gene targeting in human iPSCs will help to establish better models for human biology and disease research and to provide proof-of-principle for future gene therapy.

Human oocytes reprogram somatic cells to a pluripotent state

The exchange of the oocyte’s genome with the genome of a somatic cell, followed by the derivation of pluripotent stem cells, could enable the generation of specific cells affected in degenerative human diseases. Such cells, carrying the patient’s genome, might be useful for cell replacement. Here we report that the development of human oocytes after genome exchange arrests at late cleavage stages in association with transcriptional abnormalities. In contrast, if the oocyte genome is not removed and the somatic cell genome is merely added, the resultant triploid cells develop to the blastocyst stage. Stem cell lines derived from these blastocysts differentiate into cell types of all three germ layers, and a pluripotent gene expression program is established on the genome derived from the somatic cell. This result demonstrates the feasibility of reprogramming human cells using oocytes and identifies removal of the oocyte genome as the primary cause of developmental failure after genome exchange.

Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.

Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and their genetic material is simultaneously amplified for shotgun sequencing. MIDAS reduces amplification bias because polymerase cloning occurs in physically separated, nanoliter-scale reactors, facilitating the de novo assembly of near-complete microbial genomes from single Escherichia coli cells. In addition, MIDAS allowed us to detect single-copy number changes in primary human adult neurons at 1- to 2-Mb resolution. MIDAS can potentially further the characterization of genomic diversity in many heterogeneous cell populations.

A public resource facilitating clinical use of genomes

Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved “open consent” process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain—we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.

Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming-specific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.

The Presenilin-1 ΔE9 Mutation Results in Reduced γ-Secretase Activity, but Not Total Loss of PS1 Function, in Isogenic Human Stem Cells

Presenilin 1 (PS1) is the catalytic core of γ-secretase, which cleaves type 1 transmembrane proteins, including the amyloid precursor protein (APP). PS1 also has γ-secretase-independent functions, and dominant PS1 missense mutations are the most common cause of familial Alzheimers disease (FAD). Whether PS1 FAD mutations are gain- or loss-of-function remains controversial, primarily because most studies have relied on overexpression in mouse and/or nonneuronal systems. We used isogenic euploid human induced pluripotent stem cell lines to generate and study an allelic series of PS1 mutations, including heterozygous null mutations and homozygous and heterozygous FAD PS1 mutations. Rigorous analysis of this allelic series in differentiated, purified neurons allowed us to resolve this controversy and to conclude that FAD PS1 mutations, expressed at normal levels in the appropriate cell type, impair γ-secretase activity but do not disrupt γ-secretase-independent functions of PS1. Thus, FAD PS1 mutations do not act as simple loss of PS1 function but instead dominantly gain an activity toxic to some, but not all, PS1 functions.