Early detection has the potential to reduce cancer mortality, but an effective screening test must demonstrate asymptomatic cancer detection years before conventional diagnosis in a longitudinal ...study. In the Taizhou Longitudinal Study (TZL), 123,115 healthy subjects provided plasma samples for long-term storage and were then monitored for cancer occurrence. Here we report the preliminary results of PanSeer, a noninvasive blood test based on circulating tumor DNA methylation, on TZL plasma samples from 605 asymptomatic individuals, 191 of whom were later diagnosed with stomach, esophageal, colorectal, lung or liver cancer within four years of blood draw. We also assay plasma samples from an additional 223 cancer patients, plus 200 primary tumor and normal tissues. We show that PanSeer detects five common types of cancer in 88% (95% CI: 80-93%) of post-diagnosis patients with a specificity of 96% (95% CI: 93-98%), We also demonstrate that PanSeer detects cancer in 95% (95% CI: 89-98%) of asymptomatic individuals who were later diagnosed, though future longitudinal studies are required to confirm this result. These results demonstrate that cancer can be non-invasively detected up to four years before current standard of care.
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.
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.
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.
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► Whole-genome sequencing in autism reveals wide variation in regional mutation rates ► Regional mutability can be explained by intrinsic properties of DNA ► Dense mutation clusters can be explained by compound mutation or gene conversion ► Hypermutability is a characteristic of genes involved in human disease
Whole-genome sequencing of autism cases and controls reveals that mutation rates vary by up to 100-fold throughout the genome. Mutational hot spots may be explained by intrinsic characteristics of DNA sequence and chromatin structure and appear to be characteristic of genes involved in ASD and other diseases.
The recent finding that reprogrammed human pluripotent stem cells can be derived by nuclear transfer into human oocytes as well as by induced expression of defined factors has revitalized the debate ...on whether one approach might be advantageous over the other. Here we compare the genetic and epigenetic integrity of human nuclear-transfer embryonic stem cell (NT-ESC) lines and isogenic induced pluripotent stem cell (iPSC) lines, derived from the same somatic cell cultures of fetal, neonatal, and adult origin. The two cell types showed similar genome-wide gene expression and DNA methylation profiles. Importantly, NT-ESCs and iPSCs had comparable numbers of de novo coding mutations, but significantly more than parthenogenetic ESCs. As iPSCs, NT-ESCs displayed clone- and gene-specific aberrations in DNA methylation and allele-specific expression of imprinted genes. The occurrence of these genetic and epigenetic defects in both NT-ESCs and iPSCs suggests that they are inherent to reprogramming, regardless of derivation approach.
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•Isogenic human NT-ESCs and iPSCs were derived from the same somatic cell cultures•Human NT-ESCs and iPSCs show similar profiles of gene expression and DNA methylation•De novo coding mutations occur at the same rate in human NT-ESC and iPSC lines•Loss of imprinting occurs in both NT-ESC and iPSC lines at similar frequencies
Johannesson et al. compare human somatic cells reprogrammed to pluripotency via SCNT and as iPSCs. They find similar gene expression and DNA methylation profiles, as well as comparable levels of genomic aberrations such as coding mutations and imprinted gene expression defects. This suggests that neither reprogramming method is superior in this regard.
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 Alzheimer’s 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.
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•TALENs were used to generate iPSCs with an allelic series of PS1 mutations•FAD PS1 ΔE9 mutation increases Aβ42/Aβ40 ratio in a dose-dependent manner•FAD PS1 mutations are not equivalent to PS1 haploinsufficiency
Genome-editing technology in stem cells has now been used to generate an allelic series of presenilin-1 mutations. Goldstein and colleagues find that, when a presenilin-1 mutation linked to familial Alzheimer’s disease is expressed at endogenous levels in an isogenic background in human neurons, it is not equivalent to presenilin-1 haploinsufficiency and, therefore, does not contribute to disease via a simple loss-of-function mechanism.
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.
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.
The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the CNS may differ from those present in embryonic stem cells. Here, we ...present quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC), and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes and that, surprisingly, strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.
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► Genome-wide analysis of 5hmC, 5mC and expression in distinct CNS cell types in vivo ► 5hmC is enriched in active genes in neurons ► Identification of MeCP2 as a major binding protein for 5hmC in brain cells ► A Rett Syndrome mutation in MeCP2 preferentially inhibits 5hmC binding
MeCP2 binds not only 5mC, but also 5hmC, a cytosine derivative enriched in active genes in neurons. A Rett syndrome mutation in MeCP2 preferentially inhibits 5hmC binding, suggesting a possible involvement of MeCP2’s ability to “read” 5hmC in normal brain development.
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