A recurrent 14q32.2 microdeletion mediated by expanded TGG repeats

Nearly all recurrent microdeletion/duplication syndromes described to date are characterized by the presence of flanking low copy repeats that act as substrates for non-allelic homologous recombination (NAHR) leading to the loss, gain or disruption of dosage sensitive genes. We describe an identical 1.11 Mb heterozygous deletion of 14q32.2 including the DLK1/GTL2 imprinted gene cluster in two unrelated patients. In both patients, the deleted chromosome 14 was of paternal origin, and consistent with this both exhibit clinical features compatible with uniparental disomy (UPD) (14)mat. Using a high-resolution oligonucleotide array, we mapped the breakpoints of this recurrent deletion to large flanking (TGG)n tandem repeats, each approximately 500 bp in size and sharing ≥88% homology. These expanded (TGG)n motifs share features with known fragile sites and are predicted to form strong guanine quadruplex secondary structures. We suggest that this recurrent deletion is mediated either by NAHR between the TGG repeats, or alternatively results from their inherent instability and/or strong secondary structure. Our results define a recurrent microdeletion of the 14q32.2 imprinted gene cluster mediated by flanking (TGG)n repeats, identifying a novel mechanism of recurrent genomic rearrangement. Our observation that expanded repeats can act as catalysts for genomic rearrangement extends the role of triplet repeats in human disease, raising the possibility that similar repeat structures may act as substrates for pathogenic rearrangements genome-wid

Statistical analysis of high-dimensional biomedical data: a gentle introduction to analytical goals, common approaches and challenges

International audienceBackground: In high-dimensional data (HDD) settings, the number of variables associated with each observation is very large. Prominent examples of HDD in biomedical research include omics data with a large number of variables such as many measurements across the genome, proteome, or metabolome, as well as electronic health records data that have large numbers of variables recorded for each patient. The statistical analysis of such data requires knowledge and experience, sometimes of complex methods adapted to the respective research questions. Methods: Advances in statistical methodology and machine learning methods offer new opportunities for innovative analyses of HDD, but at the same time require a deeper understanding of some fundamental statistical concepts. Topic group TG9 “High-dimensional data” of the STRATOS (STRengthening Analytical Thinking for Observational Studies) initiative provides guidance for the analysis of observational studies, addressing particular statistical challenges and opportunities for the analysis of studies involving HDD. In this overview, we discuss key aspects of HDD analysis to provide a gentle introduction for non-statisticians and for classically trained statisticians with little experience specific to HDD. Results: The paper is organized with respect to subtopics that are most relevant for the analysis of HDD, in particular initial data analysis, exploratory data analysis, multiple testing, and prediction. For each subtopic, main analytical goals in HDD settings are outlined. For each of these goals, basic explanations for some commonly used analysis methods are provided. Situations are identified where traditional statistical methods cannot, or should not, be used in the HDD setting, or where adequate analytic tools are still lacking. Many key references are provided. Conclusions: This review aims to provide a solid statistical foundation for researchers, including statisticians and non-statisticians, who are new to research with HDD or simply want to better evaluate and understand the results of HDD analyses

Modulation of Angiogenic and Inflammatory Response in Glioblastoma by Hypoxia

Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy

A Randomized Controlled Clinical Trial in Healthy Older Adults to Determine Efficacy of Glycine and N-Acetylcysteine Supplementation on Glutathione Redox Status and Oxidative Damage.

Glycine and cysteine are non-essential amino acids that are required to generate glutathione, an intracellular tripeptide that neutralizes reactive oxygen species and prevents tissue damage. During aging glutathione demand is thought to increase, but whether additional dietary intake of glycine and cysteine contributes towards the generation of glutathione in healthy older adults is not well understood. We investigated supplementation with glycine and n-acetylcysteine (GlyNAC) at three different daily doses for 2 weeks (low dose: 2.4 g, medium dose: 4.8 g, or high dose: 7.2 g/day, 1:1 ratio) in a randomized, controlled clinical trial in 114 healthy volunteers. Despite representing a cohort of healthy older adults (age mean = 65 years), we found significantly higher baseline levels of markers of oxidative stress, including that of malondialdehyde (MDA, 0.158 vs. 0.136 µmol/L, p < 0.0001), total cysteine (Cysteine-T, 314.8 vs. 276 µM, p < 0.0001), oxidized glutathione (GSSG, 174.5 vs. 132.3 µmol/L, p < 0.0001), and a lower ratio of reduced to oxidized glutathione (GSH-F:GSSG) (11.78 vs. 15.26, p = 0.0018) compared to a young reference group (age mean = 31.7 years, n = 20). GlyNAC supplementation was safe and well tolerated by the subjects, but did not increase levels of GSH-F:GSSG (end of study, placebo = 12.49 vs. 7.2 g = 12.65, p-value = 0.739) or that of total glutathione (GSH-T) (end of study, placebo = 903.5 vs. 7.2 g = 959.6 mg/L, p-value = 0.278), the primary endpoint of the study. Post-hoc analyses revealed that a subset of subjects characterized by high oxidative stress (above the median for MDA) and low baseline GSH-T status (below the median), who received the medium and high doses of GlyNAC, presented increased glutathione generation (end of study, placebo = 819.7 vs. 4.8g/7.2 g = 905.4 mg/L, p-value = 0.016). In summary GlyNAC supplementation is safe, well tolerated, and may increase glutathione levels in older adults with high glutathione demand. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT05041179, NCT05041179

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data

Motivation: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. Availability: The R package absfilter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Altered H19/miR‐675 expression in skeletal muscle is associated with low muscle mass in community‐dwelling older adults

Background: Despite increasing knowledge of the pathogenesis of muscle ageing, the molecular mechanisms are poorly understood. Based on an expression analysis of muscle biopsies from older Caucasian men, we undertook an in-depth analysis of the expression of the long non-coding RNA, H19, to identify molecular mechanisms that may contribute to the loss of muscle mass with age. Methods: We carried out transcriptome analysis of vastus lateralis muscle biopsies from 40 healthy Caucasian men aged 68–76 years from the Hertfordshire Sarcopenia Study (HSS) with respect to appendicular lean mass adjusted for height (ALMi). Validation and replication was carried out using qRT-PCR in 130 independent male and female participants aged 73–83 years recruited into an extension of the HSS (HSSe). DNA methylation was assessed using pyrosequencing. Results: Lower ALMi was associated with higher muscle H19 expression (r2 = 0.177, P < 0.001). The microRNAs, miR-675-5p/3p encoded by exon 1 of H19, were positively correlated with H19 expression (Pearson r = 0.192 and 0.182, respectively, P < 0.03), and miR-675-5p expression negatively associated with ALMi (r2 = 0.629, P = 0.005). The methylation of CpGs within the H19 imprinting control region (ICR) were negatively correlated with H19 expression (Pearson r = −0.211 to −0.245, P ≤ 0.05). Moreover, RNA and protein levels of SMAD1 and 5, targets of miR-675-3p, were negatively associated with miR-675-3p (r2 = 0.792 and 0.760, respectively) and miR-675-5p (r2 = 0.584 and 0.723, respectively) expression, and SMAD1 and 5 RNA levels positively associated with greater type II fibre size (r2 = 0.184 and 0.246, respectively, P < 0.05). Conclusions: Increased expression profiles of H19/miR-675-5p/3p and lower expression of the anabolic SMAD1/5 effectors of bone morphogenetic protein (BMP) signalling are associated with low muscle mass in older individuals

Quantifying ChIP-seq data:A spiking method providing an internal reference for sample-to-sample normalization

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments are widely used to determine, within entire genomes, the occupancy sites of any protein of interest, including, for example, transcription factors, RNA polymerases, or histones with or without various modifications. In addition to allowing the determination of occupancy sites within one cell type and under one condition, this method allows, in principle, the establishment and comparison of occupancy maps in various cell types, tissues, and conditions. Such comparisons require, however, that samples be normalized. Widely used normalization methods that include a quantile normalization step perform well when factor occupancy varies at a subset of sites, but may miss uniform genome-wide increases or decreases in site occupancy. We describe a spike adjustment procedure (SAP) that, unlike commonly used normalization methods intervening at the analysis stage, entails an experimental step prior to immunoprecipitation. A constant, low amount from a single batch of chromatin of a foreign genome is added to the experimental chromatin. This "spike" chromatin then serves as an internal control to which the experimental signals can be adjusted. We show that the method improves similarity between replicates and reveals biological differences including global and largely uniform changes

Regulation of fibrinogen production by microRNAs

Elevated levels of fibrinogen are associated with increased risk of cardiovascular disease, whereas low fibrinogen can lead to a bleeding disorder. We investigated whether microRNAs (miRNAs), known to act as post-transcriptional regulators of gene expression, regulate fibrinogen production. Using transfection of a library of 470 annotated human miRNA precursor molecules in HuH7 hepatoma cells and quantitative measurements of fibrinogen production, we identified 23 miRNAs with down-regulating (up to 64% decrease) and 4 with up-regulating effects (up to 129% increase) on fibrinogen production. Among the down-regulating miRNAs, we investigated the mechanism of action of 3 hsa-miR-29 family members and hsa-miR-409-3p. Overexpression of hsa-miR-29 members led to decreased steady-state levels of all fibrinogen gene (FGA, FGB, and FGG) transcripts in HuH7 cells. Luciferase reporter gene assays demonstrated that this was independent of miRNA-fibrinogen 3'-untranslated region interactions. In contrast, overexpression of hsa-miR-409-3p specifically lowered fibrinogen Bbeta mRNA levels, and this effect was dependent on a target site in the fibrinogen Bbeta mRNA 3'-untranslated region. This study adds to the known mechanisms that control fibrinogen production, points toward a potential cause of variable circulating fibrinogen levels, and demonstrates that a screening approach can identify miRNAs that regulate clinically important proteins