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The POLARIX trial demonstrated the superiority of polatuzumab vedotin (Pola) over vincristine in the R-CHOP regimen for large B-cell lymphomas, but it is unknown if Pola can be safely incorporated into intensified regimens (eg. DA-EPOCH-R) typically utilized for the highest risk histologies. This was a single-center, open label, prospective clinical trial of 6 cycles of Pola-DA-EPCH-R in aggressive large B-cell lymphomas. The primary endpoint was to estimate the safety of Pola-DA-EPCH-R as measured by the rate of dose-limiting toxicities (DLTs) in the first 2 cycles with pre-specified suspension rules. Secondary and exploratory endpoints included efficacy and correlation with ctDNA levels. We enrolled 18 patients on study, and with only three DLTs observed, the study met its primary endpoint for safety. There were five serious adverse events including grade 3 febrile neutropenia (3, 17%), grade 3 colonic perforation in the setting of diverticulitis (1, 6%), and grade 5 sepsis/typhlitis (1, 6%). Among 17 evaluable patients, the best overall response rate was 100% and complete response rate of 76%. With median follow up of 12.9 months, 12-month EFS was 72% (95% CI 54-96%) and 12-month OS was 94% (95% CI 84-100%). No patient with undetectable ctDNA at the end of treatment has relapsed to date. Using Pola to replace vincristine in the DA-EPOCH-R regimen met its primary safety endpoint. These data support the further evaluation and use of this approach in histologies where the potential benefit of both an intensified regimen and Pola may be desired.

Background Noninvasive prenatal screening (NIPS) of common aneuploidies using cell-free DNA from maternal plasma is part of routine prenatal care and is widely used in both high-risk and low-risk patient populations. High specificity is needed for clinically acceptable positive predictive values. Maternal copy-number variants (mCNVs) have been reported as a source of false-positive aneuploidy results that compromises specificity. Methods We surveyed the mCNV landscape in 87,255 patients undergoing NIPS. We evaluated both previously reported and novel algorithmic strategies for mitigating the effects of mCNVs on the screen’s specificity. Further, we analyzed the frequency, length, and positional distribution of CNVs in our large dataset to investigate the curation of novel fetal microdeletions, which can be identified by NIPS but are challenging to interpret clinically. Results mCNVs are common, with 65% of expecting mothers harboring an autosomal CNV spanning more than 200 kb, underscoring the need for robust NIPS analysis strategies. By analyzing empirical and simulated data, we found that general, outlier-robust strategies reduce the rate of mCNV-caused false positives but not as appreciably as algorithms specifically designed to account for mCNVs. We demonstrate that large-scale tabulation of CNVs identified via routine NIPS could be clinically useful: together with the gene density of a putative microdeletion region, we show that the region’s relative tolerance to duplications versus deletions may aid the interpretation of microdeletion pathogenicity. Conclusions Our study thoroughly investigates a common source of NIPS false positives and demonstrates how to bypass its corrupting effects. Our findings offer insight into the interpretation of NIPS results and inform the design of NIPS algorithms suitable for use in screening in the general obstetric population. Electronic supplementary material The online version of this article (10.1186/s12920-018-0410-6) contains supplementary material, which is available to authorized users.

BackgroundHereditary cancer screening (HCS) for germline variants in the 3’ exons of PMS2, a mismatch repair gene implicated in Lynch syndrome, is technically challenging due to homology with its pseudogene PMS2CL. Sequences of PMS2 and PMS2CL are so similar that next-generation sequencing (NGS) of short fragments—common practice in multigene HCS panels—may identify the presence of a variant but fail to disambiguate whether its origin is the gene or the pseudogene. Molecular approaches utilizing longer DNA fragments, such as long-range PCR (LR-PCR), can definitively localize variants in PMS2, yet applying such testing to all samples can have logistical and economic drawbacks.MethodsTo address these drawbacks, we propose and characterize a reflex workflow for variant discovery in the 3’ exons of PMS2. We cataloged the natural variation in PMS2 and PMS2CL in 707 samples and designed hybrid-capture probes to enrich the gene and pseudogene with equal efficiency. For PMS2 exon 11, NGS reads were aligned, filtered using gene-specific variants, and subject to standard diploid variant calling. For PMS2 exons 12-15, the NGS reads were permissively aligned to PMS2, and variant calling was performed with the expectation of observing four alleles (i.e., tetraploid calling). In this reflex workflow, short-read NGS identifies potentially reportable variants that are then subject to disambiguation via LR-PCR-based testing.ResultsApplying short-read NGS screening to 299 HCS samples and cell lines demonstrated >99% analytical sensitivity and >99% analytical specificity for single-nucleotide variants (SNVs) and short insertions and deletions (indels), as well as >96% analytical sensitivity and >99% analytical specificity for copy-number variants. Importantly, 92% of samples had resolved genotypes from short-read NGS alone, with the remaining 8% requiring LR-PCR reflex.ConclusionOur reflex workflow mitigates the challenges of screening in PMS2 and serves as a guide for clinical laboratories performing multigene HCS. To facilitate future exploration and testing of PMS2 variants, we share the raw and processed LR-PCR data from commercially available cell lines, as well as variant frequencies from a diverse patient cohort.

BACKGROUND By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often METHODS We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS Validated high-fidelity identification of different variant types—especially for diseases with complicated molecular genetics—maximizes at-risk couple detection.

The PNAS paper “Recurrent rewiring and emergence of RNA regulatory networks” by Wilinski et al. (1) has a number of flaws. The paper incorrectly interprets the data underlying its major, novel claim about target rewiring. The paper also presents models that conflict with previous results and does not consider existing models that do explain all available results. First, the major novel result from Wilinski et al. (1) is that Neurospora crassa Puf4/5 targets are enriched for cytosolic ribosomal protein mRNAs and that these interactions have been conserved since the common ancestor of Saccharomycotina and Pezizomycotina. This result conflicts with published results (2) and … [↵][1]1Email: gjhogan{at}alumni.stanford.edu. [1]: #xref-corresp-1-1

PurposeBy identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the diseases’ respective prevalences, and the screen’s sensitivity for identifying disease-causing variants. Here we present an analytical validation of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide copy-number-variant (CNV) calling, and customized assays for technically challenging genes.MethodsNext-generation sequencing, a customized bioinformatics pipeline, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precludes novel variant identification or interpretation. Variant calls were compared to reference and orthogonal data.ResultsValidation of our ECS data demonstrated >99% analytical sensitivity and >99% specificity. A preliminary assessment of 15,177 patient samples reveals the substantial impact on fetal disease-risk detection attributable to novel CNV calling (13.9% of risk) and technically challenging conditions (15.5% of risk), such as congenital adrenal hyperplasia.ConclusionValidated, high-fidelity identification of different variant types—especially in diseases with complicated molecular genetics—maximizes at-risk couple detection.

The past two decades have brought many important advances in our understanding of the hereditary susceptibility to cancer. Numerous studies have provided convincing evidence that identification of germline mutations associated with hereditary cancer syndromes can lead to reductions in morbidity and mortality through targeted risk management options. Additionally, advances in gene sequencing technology now permit the development of multigene hereditary cancer testing panels. Here, we describe the 2016 revision of the Counsyl Inherited Cancer Screen for detecting single-nucleotide variants (SNVs), short insertions and deletions (indels), and copy number variants (CNVs) in 36 genes associated with an elevated risk for breast, ovarian, colorectal, gastric, endometrial, pancreatic, thyroid, prostate, melanoma, and neuroendocrine cancers. To determine test accuracy and reproducibility, we performed a rigorous analytical validation across 341 samples, including 118 cell lines and 223 patient samples. The screen achieved 100% test sensitivity across different mutation types, with high specificity and 100% concordance with conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). We also demonstrated the screen’s high intra-run and inter-run reproducibility and robust performance on blood and saliva specimens. Furthermore, we showed that pathogenic Alu element insertions can be accurately detected by our test. Overall, the validation in our clinical laboratory demonstrated the analytical performance required for collecting and reporting genetic information related to risk of developing hereditary cancers.

PurposeThe recent growth in pan-ethnic expanded carrier screening (ECS) has raised questions about how such panels might be designed and evaluated systematically. Design principles for ECS panels might improve clinical detection of at-risk couples and facilitate objective discussions of panel choice.MethodsGuided by medical-society statements, we propose a method for the design of ECS panels that aims to maximize the aggregate and per-disease sensitivity and specificity across a range of Mendelian disorders considered serious by a systematic classification scheme. We evaluated this method retrospectively using results from 474,644 de-identified carrier screens. We then constructed several idealized panels to highlight strengths and limitations of different ECS methodologies.ResultsBased on modeled fetal risks for “severe” and “profound” diseases, a commercially available ECS panel (Counsyl) is expected to detect 183 affected conceptuses per 100,000 US births. A screen’s sensitivity is greatly impacted by two factors: (1) the methodology used (e.g., full-exon sequencing finds more affected conceptuses than targeted genotyping), and (2) the detection rate of the screen for diseases with high prevalence and complex molecular genetics (e.g., fragile X syndrome).ConclusionThe described approaches allow principled, quantitative evaluation of which diseases and methodologies are appropriate for pan-ethnic expanded carrier screening.

Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.

During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28–30 nucleotides and 20–22 nucleotides long, representing translating ribosomes in distinct states, differentially stabilized by specific elongation inhibitors. We find that the balance of small and large footprints varies by codon and is correlated with translation speed. The ability to visualize conformational changes in the ribosome during elongation, at single-codon resolution, provides a new way to study the detailed kinetics of translation and a new probe with which to identify the factors that affect each step in the elongation cycle. DOI: http://dx.doi.org/10.7554/eLife.01257.001, eLife digest To make a protein from a gene, the gene is first transcribed to produce a molecule of messenger RNA (mRNA), which then passes through a molecular machine called a ribosome. The ribosome reads the genetic code in the mRNA in groups of three letters at a time, and each triplet of letters (or codon) represents an amino acid. The ribosome then joins the relevant amino acids together to build a protein. The ribosome processes about six amino acids per second, on average, but the mRNA is not fed through at a constant rate. Instead, the ribosome changes its shape to ratchet along the mRNA from one codon to the next: it then reads the new codon and adds another amino acid to the protein. However, many of the details of this ratcheting process are not fully understood. In this study, Lareau, Hite et al. have used a technique called ‘ribosome profiling’ to explore the movement of ribosomes along mRNA molecules. First, all of the pieces of mRNA molecules that are not protected inside a ribosome were chemically destroyed. The sequences of the protected fragments were then read and matched to the full-length gene sequences. The protected fragments came in two different sizes: some were about 28–30 letters long, and others were about 20–22 letters long. Lareau, Hite et al. suggest that these different fragment sizes occur because the ribosome switches between two shapes at each codon as it ratchets along the mRNA, and so it protects different lengths of mRNA. In previous ribosome-profiling experiments, the fragments had all been about 28 letters long; but these experiments had used a chemical to halt the progress of the ribosomes along the mRNAs before measuring the length of the fragments. Lareau, Hite et al. argue that this chemical locks the ribosome in the same shape when it brings the ribosome to a halt, and so the protected fragments always have the same length. Further, other chemicals that halt ribosomes appear to lock this molecular machine in the other shape, and so it can only protect the shorter fragments. The findings of Lareau, Hite et al. show that ribosomal profiling experiments can reveal much more than simply where a ribosome is on an mRNA molecule. Further study into the different stages of the ribosome ratcheting process will help uncover how the speed that a ribosome translates an mRNA into a protein can be encoded in the mRNA sequence itself. DOI: http://dx.doi.org/10.7554/eLife.01257.002

An unexpectedly large fraction of genes in metazoans (human, mouse, zebrafish, worm, fruit fly) express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant (Arabidopsis thaliana), and protists (Plasmodium falciparum and Dictyostelium discoideum). For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼ 100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or 'piggyback' on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.

Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady-state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in Saccharomyces cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation-coupled microarray studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units.

To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.