RNA-Seq Perspectives to Improve Clinical Diagnosis

Marco-Puche, Guillermo; Lois, Sergio; Benítez, Javier; Triviño, Juan Carlos

doi:10.3389/fgene.2019.01152

Cited by 81 publications

(63 citation statements)

References 64 publications

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“…The current methodology may serve as a template for the quantification of alternative splicing or of countless other mutations that bring about disease through aberrant splicing. Routine assessment of the latter, including the common thalassemic HBB IVSII−654(C > T) (HBB:c.316-197C > T) mutation [27], by conventional RT-PCR methods has limited utility, whereas assessments of splice events by targeted or global RNA-seq may impose unsuitable technical, cost or material requirements for many applications [28,29]. Adoption of the current assay would be simple and cost-effective and would allow clear conclusions on the regulation of alternative splicing or on the effect of therapeutic interventions, including pharmacological treatments, DNA editing or targeted mRNA knock-down, and on molecular mechanisms underlying their action.…”

Section: Discussionmentioning

confidence: 99%

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia

Patsali

Papasavva

Christou

et al. 2020

IJMS

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The β-thalassemias are an increasing challenge to health systems worldwide, caused by absent or reduced β-globin (HBB) production. Of particular frequency in many Western countries is HBBIVSI−110(G > A) β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the HBB gene, and while partially retaining normal splicing of HBB, it severely reduces HBB protein expression from the mutant locus and HBB loci in trans. For the assessment of the underlying mechanisms and of therapies targeting β-thalassemia, accurate quantification of aberrant and normal HBB mRNA is essential, but to date, has only been performed by approximate methods. To address this shortcoming, we have developed an accurate, duplex reverse-transcription quantitative PCR assay for the assessment of the ratio and absolute quantities of normal and aberrant mRNA species as a tool for basic and translational research of HBBIVSI−110(G > A) β-thalassemia. The method was employed here to determine mRNA ratios and quantities in blood and primary cell culture samples and correlate them with HBB protein levels. Moreover, with its immediate utility for β-thalassemia and the mutation in hand, the approach can readily be adopted for analysis of alternative splicing or for quantitative assays of any disease-causing mutation that interferes with normal splicing.

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Section: Discussionmentioning

confidence: 99%

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia

Patsali

Papasavva

Christou

et al. 2020

IJMS

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“…In clinical genetics there are still many cases where a diagnosis cannot be made 20 . For some of these undiagnosed cases, RNA-Sequencing can help make a diagnosis 21 , and outlier ASE has recently been used as diagnostic tool for muscular disease patients 22 . However, to know what effect a very rare pathogenic variant might have, ASE must be analysed in a large dataset.…”

Section: Discussionmentioning

confidence: 99%

Imbalanced expression for predicted high-impact, autosomal-dominant variants in a cohort of 3,818 healthy samples

Klein

Deelen

Urzua

et al. 2020

Preprint

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BackgroundOne of the growing problems in genome diagnostics is the increasing number of variants that get identified through genetic testing but for which it is unknown what the significance for the disease is (Variants of Unknown Significance - VUS)1,2. When these variants are observed in patients, clinicians need to be able to determine their relevance for causing the patient’s disease. Here we investigated whether allele-specific expression (ASE) can be used to prioritize disease-relevant VUS and therefore assist diagnostics. In order to do so, we conducted ASE analysis in RNA-seq data from 3,818 blood samples (part of the the Dutch BIOS biobank consortium), to ascertain how VUS affect gene expression. We compared the effect of VUS variants to variants that are predicted to have a high impact, and variants that are predicted to be pathogenic but are either recessive or autosomal-dominant with low penetrance.ResultsFor immune and haematological disorders, we observed that 24.7% of known pathogenic variants from ClinVar show allelic imbalance in blood, as compared to 6.6% of known benign variants with matching allele frequencies. However, for other types of disorders, ASE information from blood did not distinguish (likely) pathogenic variants from benign variants. Unexpectedly, we identified 5 genes (ALOX5, COMT, PRPF8, PSTPIP1 and SH3BP2) in which seven population-based samples had a predicted high impact, autosomal-dominant variant. For these genes the imbalanced expression of the major allele compensates for the lower expression of the minor allele.ConclusionsOur analysis in a large population-based gene expression cohort reveals examples of high impact, autosomal-dominant variants that are compensated for by imbalanced expression. Additionally, we observed that ASE analyses in blood are informative for predicting pathogenic variants that are associated with immune and haematological conditions. We have made all our ASE results, including many ASE calls for rare variants (MAF < 1%), available at https://molgenis15.gcc.rug.nl/.

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“…RNA sequencing (RNA-Seq) can be used to investigate differences in gene expression at a genome-wide level. RNA-Seq has the advantages of more accurate quantification, higher repeatability, a wider detection range, and more reliable analysis than other methods [18]. In addition to analyzing gene expression levels, RNA-Seq can also be used to discover new transcripts, single nucleotide polymorphisms (SNPs), and splice variants to provide information about allele-specific gene expression [23].…”

Section: Introductionmentioning

confidence: 99%

Transcriptome analysis following enterovirus 71 and coxsackievirus A16 infection in respiratory epithelial cells

Liu

et al. 2020

Arch Virol

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Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71-and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.

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RNA-Seq Perspectives to Improve Clinical Diagnosis

Cited by 81 publications

References 64 publications

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia

Imbalanced expression for predicted high-impact, autosomal-dominant variants in a cohort of 3,818 healthy samples

Transcriptome analysis following enterovirus 71 and coxsackievirus A16 infection in respiratory epithelial cells

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