Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease.

Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.

The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1).

La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes.

Divergent clonal selection dominates medulloblastoma at recurrence.

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (<5%). Whole-genome sequencing of 33 pairs of human diagnostic and post-therapy medulloblastomas demonstrated substantial genetic divergence of the dominant clone after therapy (<12% diagnostic events were retained at recurrence). In both mice and humans, the dominant clone at recurrence arose through clonal selection of a pre-existing minor clone present at diagnosis. Targeted therapy is unlikely to be effective in the absence of the target, therefore our results offer a simple, proximal, and remediable explanation for the failure of prior clinical trials of targeted therapy.

Genomic characteristics of miscarriage copy number variants.

Studies of copy number variants (CNVs) in miscarriages are rare in comparison to post-natal cases with developmental abnormalities. The overall characteristics of miscarriage CNVs (size, gene content and function) are therefore largely unexplored. Our goal was to assess and compare the characteristics of CNVs identified in 101 euploid miscarriages from four high-resolution array studies that documented both common miscarriage CNVs (i.e. CNVs found in controls from the Database of Genomic Variants, DGV) and rare miscarriage CNVs (not reported in DGV). Our miscarriage analysis included 24 rare CNVs with 93 genes, and 372 common CNVs (merged into 119 common CNV regions; CNVRs) with 354 genes. The rare and common CNVs were comparable in size (median size of ∼ 0.16 and 0.14 Mb, respectively); however, rare CNVs showed a significantly higher gene density, with 56 genes/Mb in rare and 24 genes/Mb in common CNVs (P = 0.03). Rare CNVs also had two times more genes with mouse knock-out models which were reported for 42% of rare and 19% of common CNV genes. No specific pathway enrichment was noted for 24 rare CNV genes, but common CNV genes showed significant enrichment in genes from immune-response related pathways and pregnancy/reproduction-related biological processes. Our analysis of CNVs from euploid miscarriages suggests that both rare and common CNVs could have a role in miscarriage by impacting pregnancy-related genes or pathways. Cataloguing of all CNVs and detailed description of their characteristics (e.g. gene content, genomic breakpoints) is desirable in the future for better understanding of their relevance to pregnancy loss.

Copy number variants (CNVs) analysis in a deeply phenotyped cohort of individuals with intellectual disability (ID).

BACKGROUND: DNA copy number variants (CNVs) are found in 15% of subjects with ID but their association with phenotypic abnormalities has been predominantly studied in smaller cohorts of subjects with detailed yet non-systematically categorized phenotypes, or larger cohorts (thousands of cases) with smaller number of generalized phenotypes. METHODS: We evaluated the association of de novo, familial and common CNVs detected in 78 ID subjects with phenotypic abnormalities classified using the Winter-Baraitser Dysmorphology Database (WBDD) (formerly the London Dysmorphology Database). Terminology for 34 primary (coarse) and 169 secondary (fine) phenotype features were used to categorize the abnormal phenotypes and determine the prevalence of each phenotype in patients grouped by the type of CNV they had. RESULTS: In our cohort more than 50% of cases had abnormalities in primary categories related to head (cranium, forehead, ears, eye globes, eye associated structures, nose) as well as hands and feet. The median number of primary and secondary abnormalities was 12 and 18 per subject, respectively, indicating that the cohort consisted of subjects with a high number of phenotypic abnormalities (median De Vries score for the cohort was 5). The prevalence of each phenotypic abnormality was comparable in patients with de novo or familial CNVs in comparison to those with only common CNVs, although a trend for increased frequency of cranial and forehead abnormalities was noted in subjects with rare de novo and familial CNVs. Two clusters of subjects were identified based on the prevalence of each fine phenotypic feature, with an average of 28.3 and 13.5 abnormal phenotypes/subject in the two clusters respectively (P < 0.05). CONCLUSIONS: Our study is a rare example of using standardized, deep morphologic phenotype clustering with phenotype/CNV correlation in a cohort of subjects with ID. The composition of the cohort inevitably influences the phenotype/genotype association, and our studies show that the influence of the de novo CNVs on the phenotype is less obvious in cohorts consisting of subjects with a high number of phenotypic abnormalities. The outcome of phenotype/genotype analysis also depends on the choice of phenotypes assessed and standardized phenotyping is required to minimize variability.

miRNA and miRNA target genes in copy number variations occurring in individuals with intellectual disability.

BACKGROUND: MicroRNAs (miRNAs) are a family of short, non-coding RNAs modulating expression of human protein coding genes (miRNA target genes). Their dysfunction is associated with many human diseases, including neurodevelopmental disorders. It has been recently shown that genomic copy number variations (CNVs) can cause aberrant expression of integral miRNAs and their target genes, and contribute to intellectual disability (ID). RESULTS: To better understand the CNV-miRNA relationship in ID, we investigated the prevalence and function of miRNAs and miRNA target genes in five groups of CNVs. Three groups of CNVs were from 213 probands with ID (24 de novo CNVs, 46 familial and 216 common CNVs), one group of CNVs was from a cohort of 32 cognitively normal subjects (67 CNVs) and one group of CNVs represented 40 ID related syndromic regions listed in DECIPHER (30 CNVs) which served as positive controls for CNVs causing or predisposing to ID. Our results show that 1). The number of miRNAs is significantly higher in de novo or DECIPHER CNVs than in familial or common CNV subgroups (P < 0.01). 2). miRNAs with brain related functions are more prevalent in de novo CNV groups compared to common CNV groups. 3). More miRNA target genes are found in de novo, familial and DECIPHER CNVs than in the common CNV subgroup (P < 0.05). 4). The MAPK signaling cascade is found to be enriched among the miRNA target genes from de novo and DECIPHER CNV subgroups. CONCLUSIONS: Our findings reveal an increase in miRNA and miRNA target gene content in de novo versus common CNVs in subjects with ID. Their expression profile and participation in pathways support a possible role of miRNA copy number change in cognition and/or CNV-mediated developmental delay. Systematic analysis of expression/function of miRNAs in addition to coding genes integral to CNVs could uncover new causes of ID.

Methods to extract and analyze fluid from human pluripotent stem cell-derived choroid plexus organoids.

The choroid plexus (ChP) is a highly vascularized tissue lining the ventricular space of the brain. The ChP generates cerebrospinal fluid (CSF) and forms a protective barrier in the central nervous system (CNS). Recently, a three-dimensional human pluripotent stem cell (hPSC)-derived ChP organoid model has been developed. This model generates cystic structures that are filled with a fluid resembling CSF and are surrounded by an epithelial layer expressing ependymal choroid plexus-specific markers. Here we describe a method to generate these choroid plexus organoids using a commercial kit and methods to extract the CSF-like fluid for use in downstream analysis.

RNA-seq analysis of the mantle transcriptome from Mytilus edulis during a seasonal spawning event in deep and shallow water culture sites on the northeast coast of Newfoundland, Canada.

The blue mussel (Mytilus edulis) has global commercial and ecological importance both in wild and cultured conditions. However there is a qualitative and quantitative lack of knowledge of the molecular mechanisms associated with its reproductive physiology, especially with reference to environmental interactions. Here we initiated a transcriptomic analysis (RNA-sequencing (RNA-seq)) of the mantle from both sexes sampled during a seasonal spawning event and from two culture depths (shallow-5 m; deep- 15 m). Mantle libraries were produced from 3 males and 3 females sampled from each of two shallow sites and two deep sites for a total of 12 replicate male and 12 replicate female libraries (24 total libraries). Overall a total of 2.3 billion raw 100 base reads with an average of 96.5 million reads/library were obtained and assembled into 296,118 transcripts with an average length of 568 bp. Overall, 315 transcripts from male libraries and 25 from female libraries were found to be upregulated in deep water as compared to shallow (edgeR adjusted p value ≤ 0.05). Conversely, 126 transcripts from male libraries and 135 from female libraries were found to be significantly downregulated at the same depth. Thirteen transcripts were selected for qPCR validation based on importance in reproduction, antimicrobial defense and metabolism. Of these, 9 RNA-seq identified transcripts were shown by qPCR to be differentially expressed between groups: 2 were upregulated in deep compared with shallow water (dhx38, mt-co1), 2 were upregulated for female compared with male mantle (pias2, mapkap1) and 6 genes (fndc3a, acbd3, klhl10, ccnb3, armc4, mt-co1) showed to be upregulated in males compared to females. The majority of qPCR studied transcripts were identified as involved in gamete development based on the UniProt database. This study further characterizes the importance of the mantle transcriptome during reproductive activities of M. edulis.

GenPipes: an open-source framework for distributed and scalable genomic analyses.

BACKGROUND: With the decreasing cost of sequencing and the rapid developments in genomics technologies and protocols, the need for validated bioinformatics software that enables efficient large-scale data processing is growing. FINDINGS: Here we present GenPipes, a flexible Python-based framework that facilitates the development and deployment of multi-step workflows optimized for high-performance computing clusters and the cloud. GenPipes already implements 12 validated and scalable pipelines for various genomics applications, including RNA sequencing, chromatin immunoprecipitation sequencing, DNA sequencing, methylation sequencing, Hi-C, capture Hi-C, metagenomics, and Pacific Biosciences long-read assembly. The software is available under a GPLv3 open source license and is continuously updated to follow recent advances in genomics and bioinformatics. The framework has already been configured on several servers, and a Docker image is also available to facilitate additional installations. CONCLUSIONS: GenPipes offers genomics researchers a simple method to analyze different types of data, customizable to their needs and resources, as well as the flexibility to create their own workflows.

Spatial heterogeneity in medulloblastoma.

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

An integrated pipeline for the genome-wide analysis of transcription factor binding sites from ChIP-Seq.

ChIP-Seq has become the standard method for genome-wide profiling DNA association of transcription factors. To simplify analyzing and interpreting ChIP-Seq data, which typically involves using multiple applications, we describe an integrated, open source, R-based analysis pipeline. The pipeline addresses data input, peak detection, sequence and motif analysis, visualization, and data export, and can readily be extended via other R and Bioconductor packages. Using a standard multicore computer, it can be used with datasets consisting of tens of thousands of enriched regions. We demonstrate its effectiveness on published human ChIP-Seq datasets for FOXA1, ER, CTCF and STAT1, where it detected co-occurring motifs that were consistent with the literature but not detected by other methods. Our pipeline provides the first complete set of Bioconductor tools for sequence and motif analysis of ChIP-Seq and ChIP-chip data.

Understanding the impact of 1q21.1 copy number variant.

BACKGROUND: 1q21.1 Copy Number Variant (CNV) is associated with a highly variable phenotype ranging from congenital anomalies, learning deficits/intellectual disability (ID), to a normal phenotype. Hence, the clinical significance of this CNV can be difficult to evaluate. Here we described the consequences of the 1q21.1 CNV on genome-wide gene expression and function of selected candidate genes within 1q21.1 using cell lines from clinically well described subjects. METHODS AND RESULTS: Eight subjects from 3 families were included in the study: six with a 1q21.1 deletion and two with a 1q21.1 duplication. High resolution Affymetrix 2.7M array was used to refine the 1q21.1 CNV breakpoints and exclude the presence of secondary CNVs of pathogenic relevance. Whole genome expression profiling, studied in lymphoblast cell lines (LBCs) from 5 subjects, showed enrichment of genes from 1q21.1 in the top 100 genes ranked based on correlation of expression with 1q21.1 copy number. The function of two top genes from 1q21.1, CHD1L/ALC1 and PRKAB2, was studied in detail in LBCs from a deletion and a duplication carrier. CHD1L/ALC1 is an enzyme with a role in chromatin modification and DNA damage response while PRKAB2 is a member of the AMP kinase complex, which senses and maintains systemic and cellular energy balance. The protein levels for CHD1L/ALC1 and PRKAB2 were changed in concordance with their copy number in both LBCs. A defect in chromatin remodeling was documented based on impaired decatenation (chromatid untangling) checkpoint (DCC) in both LBCs. This defect, reproduced by CHD1L/ALC1 siRNA, identifies a new role of CHD1L/ALC1 in DCC. Both LBCs also showed elevated levels of micronuclei following treatment with a Topoisomerase II inhibitor suggesting increased DNA breaks. AMP kinase function, specifically in the deletion containing LBCs, was attenuated. CONCLUSION: Our studies are unique as they show for the first time that the 1q21.1 CNV not only causes changes in the expression of its key integral genes, associated with changes at the protein level, but also results in changes in their known function, in the case of AMPK, and newly identified function such as DCC activation in the case of CHD1L/ALC1. Our results support the use of patient lymphoblasts for dissecting the functional sequelae of genes integral to CNVs in carrier cell lines, ultimately enhancing understanding of biological processes which may contribute to the clinical phenotype.