Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation

BACKGROUND: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro. RESULTS: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP. CONCLUSION: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

BACKGROUND: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. RESULTS: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. CONCLUSION: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin

Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method

BACKGROUND: Neural stem cells (NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. RESULTS: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. CONCLUSIONS: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis

Performance of a 70-mer oligonucleotide microarray for genotyping of Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni </it>is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related <it>C. jejuni </it>isolates from chicken and human infection.</p> <p>Results</p> <p>With the exception of one isolate, the microarray data clusters the isolates according to the five groups determined by PFGE. In contrast, MLST defines only three genotypes among the isolates, indicating a lower resolution. All methods show that there is no inherit difference between isolates infecting humans and chicken, suggesting a common underlying population of <it>C. jejuni</it>. We further identify regions that frequently differ between isolates, including both previously described and novel regions. Finally, we show that genes that belong to certain functional groups differ between isolates more often than expected by chance.</p> <p>Conclusion</p> <p>In this study we demonstrated the utility of 70-mer oligonucleotide microarrays for genotyping of <it>Campylobacter jejuni </it>isolates, with resolution outperforming MLST.</p

Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana

BACKGROUND: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific. RESULTS: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes. CONCLUSIONS: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21 000 Arabidopsis transcripts

Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia

IntroductionThe suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods.MethodsFor this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL.ResultsBoth the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions.DiscussionThe filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL

Mining the transcriptome - methods and applications

Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods. The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal. This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype. To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank.QC 2010092

Beyond 1 Million Genomes (B1MG) D3.3 The B1MG data analysis challenge

&lt;p&gt;Germline and tumor whole genome sequencing (WGS) have now become a standard procedure, integral to both research and clinical practices. However, the diversity in analytical approaches across laboratories remains pronounced. This diversity calls for the establishment of cohesive standards, a need that has yet to be sufficiently addressed. Presently, there exists a scarcity of comprehensive schemes designed to authenticate or set benchmarks for the effectiveness of germline and tumor WGS pipelines.&lt;/p&gt;&lt;p&gt;Addressing this gap, the European H2020 initiative 1+MG has emerged with a specific mission: to bridge the connection between genomic and health data analyses. Achieving this mission mandates a meticulous exploration of existing voids and optimal methodologies within germline and tumor WGS. This is not only crucial for enhancing the quality of outcomes but also for fostering reproducibility and engendering trust among stakeholders.&lt;/p&gt;&lt;p&gt;To achieve these objectives, the collaborative efforts of the 1+MG and B1MG projects have been mobilised. The central focus lies in the orchestration of a somatic WGS benchmarking initiative, encompassing three distinct challenges:&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Wet Lab Challenge&lt;/strong&gt;: This segment scrutinises the library preparation and sequencing stages, with an emphasis on evaluating the precision and robustness of these processes.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Full Pipeline Challenge&lt;/strong&gt;: Encompassing library preparation, sequencing, and data analysis, this challenge offers a comprehensive evaluation of the end-to-end workflow. The goal is to assess the integrity of the entire pipeline in generating reliable results.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Dry Lab Challenge&lt;/strong&gt;: The data analysis pipeline takes centre stage in this challenge, as it seeks to appraise the computational methodologies employed in deciphering and interpreting the genomic data.&lt;/p&gt;&lt;p&gt;By structuring these challenges, the 1+MG and B1MG projects have contributed significantly towards harmonising WGS practices, fostering a unified understanding of best practices, and nurturing confidence among stakeholders. This progressive approach not only ensures high-quality outcomes but also supports the critical drive for reproducibility and reliability within the realm of genomic and health data analysis.&lt;/p&gt;&lt;p&gt;To this date, the 1+MG WG4 has organised a comprehensive quality comparison for all the stages of the somatic whole genome variant calling process. As described above, we have divided the workflow into three main tasks: the wetlab, the full pipeline, and the dry lab challenges. For each of these stages, we have collected results from all the participating labs and obtained the relevant quality metrics. The comparison of results across all labs has provided the baseline for the construction of a curated dataset of somatic variants with the highest reliability. This goldset establishes the standard of quality against which individual laboratory observations are measured.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&lt;/p&gt;&lt;p&gt;The 1+MG WG4 has provided best practices for whole genome somatic variant calling through a comprehensive benchmark of quality metrics for all stages of the process. This work has also contributed to the generation of a goldset of somatic variant calls, for both small and large variants. In a larger framework, the 1+MG WG4 sets the quality requirements of genomic data for cross-border access and for personalised medicine practice.&lt;/p&gt

Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report

Abstract Background A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 + 3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. Case presentation We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naïve CD4+ and CD8+ T-cells, increased number of activated effector memory CD8+ T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient’s cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. Conclusions There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing