A multiomic characterization of the leukemia cell line REH using short- and long-read sequencing.

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6::RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-yr-old at relapse, the cell line offers a practical model for the study of pediatric B-ALL. In recent years, short- and long-read DNA and RNA sequencing have emerged as a complement to karyotyping techniques in the resolution of structural variants in an oncological context. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole-genome sequencing, IsoSeq RNA sequencing, and short-read Illumina sequencing to create a detailed genomic and transcriptomic characterization of the REH cell line. Whole-genome sequencing clarified the molecular traits of disrupted ALL-associated genes including CDKN2A, PAX5, BTG1, VPREB1, and TBL1XR1, as well as the glucocorticoid receptor NR3C1 Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.

Comparison of high-throughput single-cell RNA-seq methods for ex vivo drug screening.

Functional precision medicine (FPM) aims to optimize patient-specific drug selection based on the unique characteristics of their cancer cells. Recent advancements in high throughput ex vivo drug profiling have accelerated interest in FPM. Here, we present a proof-of-concept study for an integrated experimental system that incorporates ex vivo treatment response with a single-cell gene expression output enabling barcoding of several drug conditions in one single-cell sequencing experiment. We demonstrate this through a proof-of-concept investigation focusing on the glucocorticoid-resistant acute lymphoblastic leukemia (ALL) E/R+ Reh cell line. Three different single-cell transcriptome sequencing (scRNA-seq) approaches were evaluated, each exhibiting high cell recovery and accurate tagging of distinct drug conditions. Notably, our comprehensive analysis revealed variations in library complexity, sensitivity (gene detection), and differential gene expression detection across the methods. Despite these differences, we identified a substantial transcriptional response to fludarabine, a highly relevant drug for treating high-risk ALL, which was consistently recapitulated by all three methods. These findings highlight the potential of our integrated approach for studying drug responses at the single-cell level and emphasize the importance of method selection in scRNA-seq studies. Finally, our data encompassing 27 327 cells are freely available to extend to future scRNA-seq methodological comparisons.

Genomic, transcriptomic and epigenomic sequencing data of the B-cell leukemia cell line REH.

OBJECTIVES: The aim of this data paper is to describe a collection of 33 genomic, transcriptomic and epigenomic sequencing datasets of the B-cell acute lymphoblastic leukemia (ALL) cell line REH. REH is one of the most frequently used cell lines for functional studies of pediatric ALL, and these data provide a multi-faceted characterization of its molecular features. The datasets described herein, generated with short- and long-read sequencing technologies, can both provide insights into the complex aberrant karyotype of REH, and be used as reference datasets for sequencing data quality assessment or for methods development. DATA DESCRIPTION: This paper describes 33 datasets corresponding to 867 gigabases of raw sequencing data generated from the REH cell line. These datasets include five different approaches for whole genome sequencing (WGS) on four sequencing platforms, two RNA sequencing (RNA-seq) techniques on two different sequencing platforms, DNA methylation sequencing, and single-cell ATAC-sequencing.

The SEQC2 epigenomics quality control (EpiQC) study.

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

Refined detection and phasing of structural aberrations in pediatric acute lymphoblastic leukemia by linked-read whole-genome sequencing.

Structural chromosomal rearrangements that can lead to in-frame gene-fusions are a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic leukemia (ALL). Traditional methods such as karyotyping and FISH struggle to accurately identify and phase such large-scale chromosomal aberrations in ALL genomes. We therefore evaluated linked-read WGS for detecting chromosomal rearrangements in primary samples of from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations and structural variants in the ALL genomes. We found that biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified previously unknown structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. We conclude that linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping and FISH.

Computational and Statistical Analysis of Array-Based DNA Methylation Data.

The characterization of aberrant DNA methylation is emerging as a key part of the study of cancer development and phenotype. The technical advancements and decreasing costs of methods for high-throughput profiling of DNA methylation have brought about a high interest in the use of such methods in disease association studies. Here we discuss the principles for DNA methylation analysis using data from the Infinium DNA methylation BeadChip assays and describe the computational steps and statistical considerations going from processing of the raw array data to analysis of differential methylation. Moreover, we provide detailed guidelines on how to perform tumor subtype classification based on DNA methylation signatures.

Transcriptome sequencing in pediatric acute lymphoblastic leukemia identifies fusion genes associated with distinct DNA methylation profiles.

BACKGROUND: Structural chromosomal rearrangements that lead to expressed fusion genes are a hallmark of acute lymphoblastic leukemia (ALL). In this study, we performed transcriptome sequencing of 134 primary ALL patient samples to comprehensively detect fusion transcripts. METHODS: We combined fusion gene detection with genome-wide DNA methylation analysis, gene expression profiling, and targeted sequencing to determine molecular signatures of emerging ALL subtypes. RESULTS: We identified 64 unique fusion events distributed among 80 individual patients, of which over 50% have not previously been reported in ALL. Although the majority of the fusion genes were found only in a single patient, we identified several recurrent fusion gene families defined by promiscuous fusion gene partners, such as ETV6, RUNX1, PAX5, and ZNF384, or recurrent fusion genes, such as DUX4-IGH. Our data show that patients harboring these fusion genes displayed characteristic genome-wide DNA methylation and gene expression signatures in addition to distinct patterns in single nucleotide variants and recurrent copy number alterations. CONCLUSION: Our study delineates the fusion gene landscape in pediatric ALL, including both known and novel fusion genes, and highlights fusion gene families with shared molecular etiologies, which may provide additional information for prognosis and therapeutic options in the future.

Deep targeted sequencing in pediatric acute lymphoblastic leukemia unveils distinct mutational patterns between genetic subtypes and novel relapse-associated genes.

To characterize the mutational patterns of acute lymphoblastic leukemia (ALL) we performed deep next generation sequencing of 872 cancer genes in 172 diagnostic and 24 relapse samples from 172 pediatric ALL patients. We found an overall greater mutational burden and more driver mutations in T-cell ALL (T-ALL) patients compared to B-cell precursor ALL (BCP-ALL) patients. In addition, the majority of the mutations in T-ALL had occurred in the original leukemic clone, while most of the mutations in BCP-ALL were subclonal. BCP-ALL patients carrying any of the recurrent translocations ETV6-RUNX1, BCR-ABL or TCF3-PBX1 harbored few mutations in driver genes compared to other BCP-ALL patients. Specifically in BCP-ALL, we identified ATRX as a novel putative driver gene and uncovered an association between somatic mutations in the Notch signaling pathway at ALL diagnosis and increased risk of relapse. Furthermore, we identified EP300, ARID1A and SH2B3 as relapse-associated genes. The genes highlighted in our study were frequently involved in epigenetic regulation, associated with germline susceptibility to ALL, and present in minor subclones at diagnosis that became dominant at relapse. We observed a high degree of clonal heterogeneity and evolution between diagnosis and relapse in both BCP-ALL and T-ALL, which could have implications for the treatment efficiency.

DNA methylome analysis of acute lymphoblastic leukemia cells reveals stochastic de novo DNA methylation in CpG islands.

AIM: To identify regions of aberrant DNA methylation in acute lymphoblastic leukemia (ALL) cells of different subtypes on a genome-wide scale. MATERIALS & METHODS: Whole-genome bisulfite sequencing (WGBS) was used to determine the DNA methylation levels in cells from four pediatric ALL patients of different subtypes. The findings were confirmed by 450k DNA methylation arrays in a large patient set. RESULTS: Compared with mature B or T cells WGBS detected on average 82,000 differentially methylated regions per patient. Differentially methylated regions are enriched to CpG poor regions, active enhancers and transcriptional start sites. We also identified approximately 8000 CpG islands with variable intermediate DNA methylation that seems to occur as a result of stochastic de novo methylation. CONCLUSION: WGBS provides an unbiased view and novel insights into the DNA methylome of ALL cells.

Histological characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney.

BACKGROUND: MCT14 (SLC16A14) is an orphan member of the monocarboxylate transporter (MCT) family, also known as the SLC16 family of secondary active transmembrane transporters. Available expression data for this transporter is limited, and in this paper we aim to characterize MCT14 with respect to tissue distribution and cellular localization in mouse brain. RESULTS: Using qPCR, we found that Slc16a14 mRNA was highly abundant in mouse kidney and moderately in central nervous system, testis, uterus and liver. Using immunohistochemistry and in situ hybridization, we determined that MCT14 was highly expressed in excitatory and inhibitory neurons as well as epithelial cells in the mouse brain. The expression was exclusively localized to the soma of neurons. Furthermore, we showed with our phylogenetic analysis that MCT14 most closely relate to the aromatic amino acid- and thyroid-hormone transporters MCT8 (SLC16A2) and MCT10 (SLC16A10), in addition to the carnitine transporter MCT9 (SLC16A9). CONCLUSIONS: We provide here the first histological mapping of MCT14 in the brain and our data are consistent with the hypothesis that MCT14 is a neuronal aromatic-amino-acid transporter.

The mutational landscape in pediatric acute lymphoblastic leukemia deciphered by whole genome sequencing.

Genomic characterization of pediatric acute lymphoblastic leukemia (ALL) has identified distinct patterns of genes and pathways altered in patients with well-defined genetic aberrations. To extend the spectrum of known somatic variants in ALL, we performed whole genome and transcriptome sequencing of three B-cell precursor patients, of which one carried the t(12;21)ETV6-RUNX1 translocation and two lacked a known primary genetic aberration, and one T-ALL patient. We found that each patient had a unique genome, with a combination of well-known and previously undetected genomic aberrations. By targeted sequencing in 168 patients, we identified KMT2D and KIF1B as novel putative driver genes. We also identified a putative regulatory non-coding variant that coincided with overexpression of the growth factor MDK. Our results contribute to an increased understanding of the biological mechanisms that lead to ALL and suggest that regulatory variants may be more important for cancer development than recognized to date. The heterogeneity of the genetic aberrations in ALL renders whole genome sequencing particularly well suited for analysis of somatic variants in both research and diagnostic applications.

PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells.

PAT4, the fourth member of the SLC36/proton dependent amino acid transporter (PAT) family, is a high-affinity, low capacity electroneutral transporter of neutral amino acids like proline and tryptophan. It has also been associated with the function of mTORC1, a complex in the mammalian target of rapamycin (mTOR) pathway. We performed in situ hybridization and immunohistological analysis to determine the expression profile of PAT4, as well as an RT-PCR study on tissue from mice exposed to leucine. We performed a phylogenetic analysis to determine the evolutionary origin of PAT4. The in situ hybridization and the immunohistochemistry on mouse brain sections and hypothalamic cells showed abundant PAT4 expression in the mouse brain intracellularly in both inhibitory and excitatory neurons, partially co-localizing with lysosomal markers and epithelial cells lining the ventricles. Its location in epithelial cells around the ventricles indicates a transport of substrates across the blood brain barrier. Phylogenetic analysis showed that PAT4 belongs to an evolutionary old family most likely predating animals, and PAT4 is the oldest member of that family.

Installation of mechanical ventilation in a horse stable: effects on air quality and human and equine airways.

OBJECTIVES: To examine the effects of installing a mechanical ventilation system at a riding-school stable on indoor air quality and human and horse airways. METHODS: The intervention was the installation of mechanical ventilation in a riding-school stable. Carbon dioxide (CO2), ammonia, particles, horse allergen, microorganisms and endotoxins were measured in the stable. The stable-workers and riding-students completed a questionnaire and underwent the following tests: analysis of nasal lavage for inflammation biomarkers; levels of exhaled nitrogen oxide (NO); measurements of daily peak-expiratory flow (PEF). The horses were examined clinically by airway endoscopy and bronchoalveolar lavage (BAL) and were analysed for cytology and biomarkers. RESULTS: Levels of CO2 were nearly halved and airborne horse allergen levels were markedly reduced (5-0.8 kU/m3) after the intervention. A decreased level of ultrafine particles was observed (8000-5400 particles/cm3) after the intervention, while total and respirable dust levels were mainly unchanged (200 and 130 µg/m3). Levels of microorganisms in surface samples decreased following the intervention, whereas airborne microorganisms and endotoxin increased. There was no significant change in human symptoms, PEF-variability, exhaled NO or inflammatory biomarkers in the nasal lavage. In horses, the mean score of lower airway mucus was significantly reduced together with the mean level of expression of interleukin-6 mRNA in BAL cells after the intervention. CONCLUSIONS: The installation of a mechanical ventilation system resulted in an increased air exchange rate, as demonstrated by reduced levels of CO2, ammonia, ultrafine particles and horse allergen. There was no significant clinical effect on human airways, but there was a tendency for reduced inflammation markers. The results on the horses may indicate less impact on their airways after the intervention.

Markers of respiratory inflammation in horses in relation to seasonal changes in air quality in a conventional racing stable.

Airborne factors in a conventionally managed racing stable and markers of pulmonary inflammation in the stabled horses were investigated on 3 occasions at 6-month intervals, including 2 winter periods and the intervening summer period. The stable measurements included inside and outside ambient temperature and relative humidity, levels of total and respirable dust, endotoxin, and 1,3-beta-glucan. Horses (n = 12) were examined in detail clinically as well as by endoscopy, bronchoalveolar lavage (BAL) cytology, and real-time polymerase chain reaction (RT-PCR) analysis of BAL-cells for IL-6 and IL-10 mRNA. Indoor measurements showed low dust levels irrespective of season. Inhalable dust, as well as 1,3-beta-glucan, were higher during the winter stabling period, whereas endotoxin levels were higher during summer. Complete data from all sampling occasions to be used for further evaluation was obtained for only 8 of the horses. There was a trend for elevation of BAL neutrophils in the horses during winter stabling that coincided with a 3.7-fold increased expression of IL-6 mRNA in BAL cells (P = 0.014). Compared to summer sampling, IL-10 mRNA expression was significantly upregulated in only 1 of the winter sampling occasions, implicating influence on immune regulation by factor/s apart from seasonal differences. Our findings suggest up-regulation of innate immunity in the airways of stabled horses; in particular involving IL-6 in association with mild elevations in respirable dust, 1,3-beta-glucan, and/or cold ambient air. However, given that this study was observational, other unmeasured environmental factors associated with winter stabling need to be considered.

Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-alpha in endobronchial biopsies in horses with recurrent airway obstruction.

BACKGROUND: The aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-alpha, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission. RESULTS: Despite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls. In comparing real-time PCR analysis to results of immunohistochemistry only IL-10 mRNA and protein levels in RAO horses on pasture were significantly correlated (rs = 0.893, p = 0.007). Curiously, in controls examined on pasture the TNF-alpha protein level was positively correlated to IL-10 mRNA expression (rs = 0.967, p = 0.007) and negatively correlated to IL-6 mRNA expression (rs = -0.971, p = 0.001). CONCLUSION: Given the complementary relationship of assessing cytokines directly by immunohistochemistry, or indirectly by PCR to mRNA, the lack of significant changes in either mRNA or protein levels of IL-6, IL-10 or TNF-alpha mRNA in RAO horses in exacerbation suggests that these particular cytokines in bronchial tissue may not play a substantive role in the active inflammation of this disease. To support this contention further studies examining time dependency of expression of IL-6, IL-10 or TNF-alpha are needed, as is expansion of the range of cytokines to include other key regulators of airway inflammation.

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome.

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.