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Summary Understanding the diversity of human tissues is fundamental to disease and requires linking genetic information, which is identical in most of an individual’s cells, with epigenetic mechanisms that could play tissue-specific roles. Surveys of DNA methylation in human tissues have established a complex landscape including both tissue-specific and invariant methylation patterns1,2. Here we report high coverage methylomes that catalogue cytosine methylation in all contexts for the major human organ systems, integrated with matched transcriptomes and genomic sequence. By combining these diverse data types with each individuals’ phased genome3, we identified widespread tissue-specific differential CG methylation (mCG), partially methylated domains, allele-specific methylation and transcription, and the unexpected presence of non-CG methylation (mCH) in almost all human tissues. mCH correlated with tissue-specific functions, and using this mark, we made novel predictions of genes that escape X-chromosome inactivation in specific tissues. Overall, DNA methylation in multiple genomic contexts varies substantially among human tissues.

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

Background: ctDNA has the potential to identify patients (pts) with early stage cancer; however, current assays are challenged by limited sensitivity (~50%), reliance on a single analyte (e.g. somatic mutation detection), and/or the need for tumor tissue or genomic DNA sequencing to interpret ctDNA results. Recent studies have demonstrated that ctDNA can be detected using other biomarkers including DNA methylation. We developed a technology in which both somatic mutations and epigenomic alterations can be analyzed in a single assay. Methods: Using a large database of cell-free DNA (cfDNA) profiles generated from advanced cancer patients, we designed a targeted sequencing assay that detects somatic variants, methylation alterations, and other epigenomic variations at transcription factor binding sites associated with CRC. Total cfDNA was extracted, partitioned based on methylation level, and analyzed. Data were then filtered using a variant classifier to differentiate tumor- from non-tumor-derived alterations without a priori knowledge of tissue or germline sequencing results. A machine learning model was trained on 111 cfDNA samples from 38 late stage and 10 early stage CRC pts and 63 age-matched cancer-free controls. For the independent test set, plasma samples (4-5mL) were collected from 72 pts with stage I-IV CRC prior to and 4 weeks after (N = 50, total of 122 samples) surgical resection. 35 age-matched cancer-free controls were similarly analyzed in the test set. Results: Of the 72 pts, 62.5% were male, and median age at CRC diagnosis was 61.5 years (range 36-85). Stage distribution was 52.8% stage I/II, 40.3% stage III, and 6.9% stage IV. In the 50 pts with post-surgical samples, clinical follow-up was available for 49 (median post-surgery follow-up: 314 days; range 15-472). Utilizing this assay, pre-surgery ctDNA detection rate was 94% (68/72); 97% in stage I/II, 90% in stage III, and 100% in stage IV. Epigenomic analysis significantly enhanced ctDNA detection relative to somatic mutational analysis alone (94% vs. 56%; p Discussion: Utilizing a plasma-only sequencing assay incorporating somatic genomic variant detection, epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants, ctDNA detection rate in early stage CRC (I-III) is 94% (63/67; 95% confidence interval 86%;98%) with 94% specificity, far outperforming the detection rate of somatic sequence variant detection alone. Clinical follow-up is ongoing to evaluate post-surgery ctDNA detection rate and disease recurrence. These results have significant implications for the clinical utility of ctDNA in early stage cancer management. Citation Format: Seung-Tae Kim, Victoria M. Raymond, Joon Oh Park, Elena Zotenko, Young Suk Park, Matthew Schultz, Won Ki Kang, Oscar Westesson, Hee-Cheol Kim, Yupeng He, Justin I. Odegaard, Stefanie A. Mortimer, William J. Greenleaf, Ariel Jaimovich, Jeeyun Lee, AmirAli Talasaz. Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early-stage colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 916.

Comparisons within expanding sequence databases have revealed a dynamic interplay among genomic and epigenomic forces in driving plant evolution. Such forces are especially obvious within the F-Box (FBX) superfamily, one of the largest and most polymorphic gene families in land plants, where its frequent lineage-specific expansions and contractions provide an excellent model to assess how genetic variation impacted gene function before and after speciation. Previous phylogenetic comparisons based on orthology, diversity, and expression patterns identified three plant FBX groups--Common, Lineage-Specific, and Pseudo(genized)--whose emergences are consistent with genomic drift evolution. Here, we examined this variance within Arabidopsis thaliana by evaluating SNPs for all 877 FBX loci from 432 naturally occurring accessions and their relationships to variations in natural selection, expression, and DNA/histone methylation. In line with their phenotypic importance, Common FBX loci have low polymorphism but high deleterious mutation rates indicative of stringent functional constraints. In contrast, the Lineage-Specific and Pseudo groups are enriched in genes with basal expression and higher SNP density and more correlated with methylation marks (RNA-directed DNA methylation and histone H3K27 trimethylation) that promote transcriptional silencing. Taken together, we propose that reversible epigenomic modifications helped shape FBX gene evolution by transcriptionally suppressing the adverse effects of gene dosage imbalance and harmful FBX alleles that arise during genomic drift, while simultaneously allowing innovations to emerge through epigenomic reprogramming.

Natural epigenetic variation provides a source for the generation of phenotypic diversity, but to understand its contribution to such diversity, its interaction with genetic variation requires further investigation. Here we report population-wide DNA sequencing of genomes, transcriptomes and methylomes of wild Arabidopsis thaliana accessions. Single cytosine methylation polymorphisms are not linked to genotype. However, the rate of linkage disequilibrium decay amongst differentially methylated regions targeted by RNA-directed DNA methylation is similar to the rate for single nucleotide polymorphisms. Association analyses of these RNA-directed DNA methylation regions with genetic variants identified thousands of methylation quantitative trait loci, which revealed the population estimate of genetically dependent methylation variation. Analysis of invariably methylated transposons and genes across this population indicates that loci targeted by RNA-directed DNA methylation are epigenetically activated in pollen and seeds, which facilitates proper development of these structures.

Summary Detailed molecular profiling of Oryza sativa (rice) was carried out to uncover the features that are essential for germination and early seedling growth under anoxic conditions. Temporal analysis of the transcriptome and methylome from germination to young seedlings under aerobic and anaerobic conditions revealed 82% similarity in the transcriptome and no differences in the epigenome up to 24 h. Following germination, significant changes in the transcriptome and DNA methylation were observed between 4-day aerobically and anaerobically grown coleoptiles. A link between the epigenomic state and cell division versus cell elongation is suggested, as no differences in DNA methylation were observed between 24-h aerobically and anaerobically germinating embryos, when there is little cell division. After that, epigenetic changes appear to correlate with differences between cell elongation (anaerobic conditions) versus cell division (aerobic conditions) in the coleoptiles. Re-oxygenation of 3-day anaerobically grown seedlings resulted in rapid transcriptomic changes in DNA methylation in these coleoptiles. Unlike the transcriptome, changes in DNA methylation upon re-oxygenation did not reflect those seen in aerobic coleoptiles, but instead, reverted to a pattern similar to dry seeds. Reversion to the ‘dry seed’ state of DNA methylation upon re-oxygenation may act to ‘reset the clock’ for the rapid molecular changes and cell division that result upon re-oxygenation.

Eukaryotes possess several RNA surveillance mechanisms that prevent undesirable aberrant RNAs from accumulating. Arabidopsis XRN2, XRN3, and XRN4 are three orthologs of the yeast 5′-to-3′ exoribonuclease, Rat1/Xrn2, that function in multiple RNA decay pathways. XRN activity is maintained by FIERY1 (FRY1), which converts the XRN inhibitor, adenosine 3′, 5′-bisphosphate (PAP), into 5′AMP. To identify the roles of XRNs and FRY1 in suppression of non-coding RNAs, strand-specific genome-wide tiling arrays and deep strand-specific RNA-Seq analyses were carried out in fry1 and xrn single and double mutants. In fry1-6, about 2000 new transcripts were identified that extended the 3′ end of specific mRNAs; many of these were also observed in genotypes that possess the xrn3-3 mutation, a partial loss-of-function allele. Mutations in XRN2 and XRN4 in combination with xrn3-3 revealed only a minor effect on 3′ extensions, indicating that these genes may be partially redundant with XRN3. We also observed the accumulation of 3′ remnants of many DCL1-processed microRNA (miRNA) precursors in fry1-6 and xrn3-3. These findings suggest that XRN3, in combination with FRY1, is required to prevent the accumulation of 3′ extensions that arise from thousands of mRNA and miRNA precursor transcripts.

Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but their low incorporation efficiency, which is possibly due to competition from release factors, limits the power and scope of this technology. Here we show that the reportedly essential release factor 1 (RF1) can be knocked out from Escherichia coli by 'fixing' release factor 2 (RF2). The resultant strain JX33 is stable and independent, and it allows UAG to be reassigned from a stop signal to an amino acid when a UAG-decoding tRNA-synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving new protein functions by enabling Uaa incorporation at multiple sites.

Bogdanovic, Ozren et al., The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases., Spanish and Andalusian government grants BFU2013-41322-P and BIO-396 to J.L.G.-S. supported this work. R.L. was supported by an Australian Research Council Future Fellowship (FT120100862) and a Sylvia and Charles Viertel Senior Medical Research Fellowship, and work in the laboratory of R.L. was funded by the Australian Research Council, National Health and Medical Research Council, and the Raine Medical Research Foundation. O.B. is supported by an Australian Research Council Discovery Early Career Researcher Award (DECRA; DE140101962). The laboratory of M.V. is supported by grants from the Netherlands Organisation for Scientific Research (NWO-VIDI; 864.09.003) and Cancer Genomics Netherlands, a European Research Council starting grant (309384) and the European Union Framework Programme 7 Network of Excellence EpiGeneSys. J.R.E. was supported by the Gordon and Betty Moore Foundation (GBMF3034) and is an Investigator of the Howard Hughes Medical Institute. Work in the laboratory of M.M. is funded by grants from the Ministerio de Economia y Competitividad (BFU2011-23083), Comunidad Autónoma de Madrid (CELLDD-CM), and by the Pro-CNIC Foundation. This work has been supported by a grant from the US National Institutes of Health (National Institute of Child Health and Human Development, grant R01HD069344) to G.J.C.V.

Different neurodegenerative disorders often show similar lesions, such as the presence of amyloid plaques, TAU-neurotangles and synuclein inclusions. The genetically inherited forms are rare, so we wondered whether shared epigenetic aberrations, such as those affecting DNA methylation, might also exist. The studied samples were gray matter samples from the prefrontal cortex of control and neurodegenerative disease-associated cases. We performed the DNA methylation analyses of Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease and Alzheimer-like neurodegenerative profile associated with Down's syndrome samples. The DNA methylation landscapes obtained show that neurodegenerative diseases share similar aberrant CpG methylation shifts targeting a defined gene set. Our findings suggest that neurodegenerative disorders might have similar pathogenetic mechanisms that subsequently evolve into different clinical entities. The identified aberrant DNA methylation changes can be used as biomarkers of the disorders and as potential new targets for the development of new therapies., Human Frontiers Science Program (HFSP) Ref. RGP0018/2007-C, the European Community’s RGP0018/2007-C (FP7/2007-2013) under grant agreement PITN-GA-2009-238242—DISCHROM project and grant agreement 278486-DEVELAGE, the Instituto de Salud Carlos III—Ministerio de Sanidad y Consumo Proyecto FIS (Fondo Investigación Sanitaria, Spain) through the E-RARE EuroRETT network, Grants P50-AG16573, HD065160-01 and SAF2007-6-8, the Fondation Lejeune, Botin Foundation, Fundació Agrupació Premi Àmbit Gent Grant and the Cellex Foundation (Catalonia

Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress. DOI: http://dx.doi.org/10.7554/eLife.09343.001, eLife digest Phosphate is an important nutrient for all living organisms. This chemical group forms part of the backbone of DNA molecules, and has a crucial role in many chemical reactions that occur inside cells. Plants in particular need a source of phosphate to grow. This is why agricultural fertilizers are rich in phosphate, but unfortunately, the use of fertilizers is not sustainable. Many researchers are now looking for new ways to maintain high crop yields without chemical fertilizers, and understanding how crops are affected in times of shortage will be pivotal to achieving this goal. DNA contains coded information in the form of genes, which can either be switched on or off. Chemical marks added to the DNA can earmark genes for activation or inactivation, a bit like handwritten annotations in an instruction manual. One example is the addition of a chemical tag called a methyl group to one of the letters of the DNA code—so-called ‘cytosine methylation’. However, little is known about how the pattern of these chemical marks on DNA changes in response to changes in the environment. Secco et al. investigated changes in cytosine methylation in both rice and Arabidopsis plants that had been deprived of phosphate. Arabidopsis, or thale cress, is a model plant that is often studied by plant biologists because it is small and grows quickly. The experiments showed that when rice plants were not given enough phosphate, the pattern of DNA methylation changed. This was particularly true around certain genes that help the plants survive in these difficult conditions. Notably, in the absence of phosphate, methylation also occurs more often in DNA sequences called transposable elements that sit close to these useful genes, and less often around other genes. Transposable elements, also known as ‘jumping genes’ can move within the genome and thus potentially have damaging effects through altering the DNA sequence. However, DNA methylation normally prevents this from happening. Therefore, the extra methylation observed by Secco et al. may be a cautionary measure to inactivate these transposable elements and limit their potential deleterious effects. Further experiments went on to show that these useful genes seem to be switched on before the DNA of these transposable elements is methylated, implying that the extra methylation observed in these transposable elements is a consequence of the activation of these nearby useful genes. By contrast, similar experiments performed in Arabidopsis reveal a very limited change in DNA methylation when the plants are grown under stressful conditions. This might be because Arabidopsis has considerably fewer transposable elements than rice. The next challenge will be to explore how significant the environmentally induced silencing of transposable elements is to the stress responses and genome integrity of crop plants. DOI: http://dx.doi.org/10.7554/eLife.09343.002

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease., National Human Genome Research Institute (U.S.) (RC1HG005334), National Human Genome Research Institute (U.S.) (R01HG004037), National Human Genome Research Institute (U.S.) (R01HG004037-S1), National Human Genome Research Institute (U.S.) (RO1NS078839), National Science Foundation (U.S.) (CAREER Award 1254200)

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human., National Science Foundation (U.S.). Graduate Research Fellowship Program, Whitehead Institute for Biomedical Research (Jerome and Florence Brill Graduate Student Fellowship), National Institutes of Health (U.S.) (grants HD 045022), National Institutes of Health (U.S.) (R37CA084198), Gordon and Betty Moore Foundation (GMBF3034)

SummaryEpigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Over the past few years the cost of DNA sequencing has plummeted while the numbers and lengths of sequencing reads have increased. This sequencing revolution has led to widespread adoption of methods to investigate genome-wide patterns of DNA methylation, collectively referred to as whole-genome bisulfite sequencing (WGBS). Single-base resolution DNA methylomes are now routinely being decoded by combining high-throughput sequencing with sodium bisulfite conversion, the gold-standard method for the detection of cytosine DNA methylation [1,2]. With increasing acquisition and analysis of DNA methylomes there is a growing need to reach a consensus on the definition(s) of the amount of methylation at a specific cytosine or region. ‘Methylation level’ is often poorly defined, and can vary significantly depending on the experimenter and the queries being addressed. Therefore, we propose a set of guidelines to be considered when analyzing ‘methylation levels’ from WGBS data.

Recoding a stop codon to an amino acid may afford orthogonal genetic systems for biosynthesizing new protein and organism properties. Although reassignment of stop codons has been found in extant organisms, a model organism is lacking to investigate the reassignment process and to direct code evolution. Complete reassignment of a stop codon is precluded by release factors (RFs), which recognize stop codons to terminate translation. Here we discovered that RF1 could be unconditionally knocked out from various Escherichia coli stains, demonstrating that the reportedly essential RF1 is generally dispensable for the E. coli species. The apparent essentiality of RF1 was found to be caused by the inefficiency of a mutant RF2 in terminating all UAA stop codons; a wild type RF2 was sufficient for RF1 knockout. The RF1-knockout strains were autonomous and unambiguously reassigned UAG to encode natural or unnatural amino acids (Uaas) at multiple sites, affording a previously unavailable model for studying code evolution and a unique host for exploiting Uaas to evolve new biological functions.

Epigenetic information, which may affect an organism’s phenotype, can be stored and stably inherited in the form of cytosine DNA methylation. Changes in DNA methylation can produce meiotically stable epialleles that affect transcription and morphology, but the rates of spontaneous gain or loss of DNA methylation are unknown. We examined spontaneously occurring variation in DNA methylation in Arabidopsis thaliana plants propagated by single-seed descent for 30 generations. We identified 114,287 CG single methylation polymorphisms and 2485 CG differentially methylated regions (DMRs), both of which show patterns of divergence compared with the ancestral state. Thus, transgenerational epigenetic variation in DNA methylation may generate new allelic states that alter transcription, providing a mechanism for phenotypic diversity in the absence of genetic mutation.

Motivation: In complex disorders, independently evolving locus pairs might interact to confer disease susceptibility, with only a modest effect at each locus. With genome-wide association studies on large cohorts, testing all pairs for interaction confers a heavy computational burden, and a loss of power due to large Bonferroni-like corrections. Correspondingly, limiting the tests to pairs that show marginal effect at either locus, also has reduced power. Here, we describe an algorithm that discovers interacting locus pairs without explicitly testing all pairs, or requiring a marginal effect at each locus. The central idea is a mathematical transformation that maps ‘statistical correlation between locus pairs’ to ‘distance between two points in a Euclidean space’. This enables the use of geometric properties to identify proximal points (correlated locus pairs), without testing each pair explicitly. For large datasets (∼106 SNPs), this reduces the number of tests from 1012 to 106, significantly reducing the computational burden, without loss of power. The speed of the test allows for correction using permutation-based tests. The algorithm is encoded in a tool called Rapid (RApid Pair IDentification) for identifying paired interactions in case–control GWAS. Results: We validated Rapid with extensive tests on simulated and real datasets. On simulated models of interaction, Rapid easily identified pairs with small marginal effects. On the benchmark disease, datasets from The Wellcome Trust Case Control Consortium, Rapid ran in about 1 CPU-hour per dataset, and identified many significant interactions. In many cases, the interacting loci were known to be important for the disease, but were not individually associated in the genome-wide scan. Availability: http://bix.ucsd.edu/projects/rapid Contact: vbafna@cs.ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Background: The safety of psoralen plus ultraviolet A (PUVA) light therapy has been an issue of debate. A few multiple-center cooperative studies have reported an increase of basal cell and squamous cell carcinomas among PUVA-treated patients. In our institute, more than 1000 patients have been treated with PUVA since 1975. Objective: We investigated the incidence of skin cancer among patients who received high doses of PUVA to see whether such incidence increased. Methods: This is a historical cohort study of two comparison groups of patients. Subjects under study were 492 psoriasis patients who received PUVA treatments between 1975 and 1989. One group of 103 patients, defined as the high-dose group, received an accumulated PUVA dose of 1000 joules/cm 2 or more; another group of 389 patients, as the low-dose group, received 200 joules/cm 2 or less. The occurrence of skin cancer in the two comparison groups is analyzed. Results: In the high-dose group we observed an increased number of patients with squamous cell carcinoma, keratoacanthoma, and actinic keratosis. We did not see any patients with genital cancer, melanoma, or an increased number of patients with basal cell carcinoma. Conclusion: The risk of squamous cell carcinoma developing in patients who received a high dose of PUVA is confirmed. We speculate a combination of factors, including PUVA, may contribute to this risk.

Nature 523, 212–216 (2015); doi:10.1038/nature14465 The y-axis label in Fig. 3f of this Letter should read ‘[mCAC/CAC]/[mCAG/CAG]’ rather than ‘[mCAC/CAC]/[mCAC/CAG]’. In addition, in the description of Fig. 4c in the main text, ‘tissue’ and ‘individual’ were swapped; it should read, “Of the ASM events that varied, 4.

This article explores the potential for discovering pretrial, or revealing during the cross-examination of an expert during trial, otherwise protected work-product materials that have been supplied to the expert witness by counsel. The first part of this article discusses the disclosure of fact work-product relied on by the expert in forming an opinion during cross-examination as well as whether it is subject to pretrial discovery. The article then analyzes whether additional protection is given to opinion work product supplied to or relied on by the expert witness. Finally, the article discusses whether the work product protection has been waived if the expert uses work product material to refresh recollection both prior to and during the trial.