Extracellular vesicles produced in B cells deliver tumor suppressor miR-335 to breast cancer cells disrupting oncogenic programming in vitro and in vivo.

The successful implementation of miRNA (miR) therapies in humans will ultimately rely on the use of vehicles with improved cellular delivery capability. Here we tested a new system that leverages extracellular vesicles (EVs) laden with a tumor suppressor miRNA (miR-335) produced in B cells by plasmid DNA induction (iEVs). We demonstrate that iEVs-335 efficiently and durably restored the endogenous miR-335 pool in human triple negative breast cancer cells, downregulated the expression of the miR-335 target gene SOX4 transcription factor, and markedly inhibited tumor growth in vivo. Remarkably, iEVs-335 mediated transcriptional effects that persisted in tumors after 60 days post orthotopic implantation. Genome-wide RNASeq analysis of cancer cells treated in vitro with iEVs-335 showed the regulation of a discrete number of genes only, without broad transcriptome perturbations. This new technology may be ideally suited for therapies aimed to restore tumor suppressor miRNAs in cancer cells, disrupting the oncogenic program established after escape from miRNA control

Genetic ancestry of participants in the National Children's Study.

BackgroundThe National Children's Study (NCS) is a prospective epidemiological study in the USA tasked with identifying a nationally representative sample of 100,000 children, and following them from their gestation until they are 21 years of age. The objective of the study is to measure environmental and genetic influences on growth, development, and health. Determination of the ancestry of these NCS participants is important for assessing the diversity of study participants and for examining the effect of ancestry on various health outcomes.ResultsWe estimated the genetic ancestry of a convenience sample of 641 parents enrolled at the 7 original NCS Vanguard sites, by analyzing 30,000 markers on exome arrays, using the 1000 Genomes Project superpopulations as reference populations, and compared this with the measures of self-reported ethnicity and race. For 99% of the individuals, self-reported ethnicity and race agreed with the predicted superpopulation. NCS individuals self-reporting as Asian had genetic ancestry of either South Asian or East Asian groups, while those reporting as either Hispanic White or Hispanic Other had similar genetic ancestry. Of the 33 individuals who self-reported as Multiracial or Non-Hispanic Other, 33% matched the South Asian or East Asian groups, while these groups represented only 4.4% of the other reported categories.ConclusionsOur data suggest that self-reported ethnicity and race have some limitations in accurately capturing Hispanic and South Asian populations. Overall, however, our data indicate that despite the complexity of the US population, individuals know their ancestral origins, and that self-reported ethnicity and race is a reliable indicator of genetic ancestry

Consanguinity and rare mutations outside of MCCC genes underlie nonspecific phenotypes of MCCD.

Purpose3-Methylcrotonyl-CoA carboxylase deficiency (MCCD) is an autosomal recessive disorder of leucine catabolism that has a highly variable clinical phenotype, ranging from acute metabolic acidosis to nonspecific symptoms such as developmental delay, failure to thrive, hemiparesis, muscular hypotonia, and multiple sclerosis. Implementation of newborn screening for MCCD has resulted in broadening the range of phenotypic expression to include asymptomatic adults. The purpose of this study was to identify factors underlying the varying phenotypes of MCCD.MethodsWe performed exome sequencing on DNA from 33 cases and 108 healthy controls. We examined these data for associations between either MCC mutational status, genetic ancestry, or consanguinity and the absence or presence/specificity of clinical symptoms in MCCD cases.ResultsWe determined that individuals with nonspecific clinical phenotypes are highly inbred compared with cases that are asymptomatic and healthy controls. For 5 of these 10 individuals, we discovered a homozygous damaging mutation in a disease gene that is likely to underlie their nonspecific clinical phenotypes previously attributed to MCCD.ConclusionOur study shows that nonspecific phenotypes attributed to MCCD are associated with consanguinity and are likely not due to mutations in the MCC enzyme but result from rare homozygous mutations in other disease genes.Genet Med 17 8, 660-667

TCF1 and LEF1 Control Treg Competitive Survival and Tfr Development to Prevent Autoimmune Diseases.

CD4+ Foxp3+ T regulatory (Treg) cells are key players in preventing lethal autoimmunity. Tregs undertake differentiation processes and acquire diverse functional properties. However, how Treg's differentiation and functional specification are regulated remains incompletely understood. Here, we report that gradient expression of TCF1 and LEF1 distinguishes Tregs into three distinct subpopulations, particularly highlighting a subset of activated Treg (aTreg) cells. Treg-specific ablation of TCF1 and LEF1 renders the mice susceptible to systemic autoimmunity. TCF1 and LEF1 are dispensable for Treg's suppressive capacity but essential for maintaining a normal aTreg pool and promoting Treg's competitive survival. As a consequence, the development of T follicular regulatory (Tfr) cells, which are a subset of aTreg, is abolished in TCF1/LEF1-conditional knockout mice, leading to unrestrained T follicular helper (Tfh) and germinal center B cell responses. Thus, TCF1 and LEF1 act redundantly to control the maintenance and functional specification of Treg subsets to prevent autoimmunity

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution

Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing

Methylome-wide Analysis of Chronic HIV Infection Reveals Five-Year Increase in Biological Age and Epigenetic Targeting of HLA

HIV-infected individuals are living longer on antiretro-viral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV- individuals. First,we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across \u3e80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA).We find that decreased HLA methylation is predictive of lower CD4/CD8T cell ratio, linking molecular aging, epigenetic regulation, and disease progression

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction

Background Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. Results We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. Conclusions These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them. Keywords: RNAseqr; RNA depletion; Illumina; NGS; ABRF; TranscriptomicsNational Cancer Institute (U.S.) (Grant P30-CA14051)National Institute of Environmental Health Sciences (Grant P30-ES002109

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.

Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines