Development and validation of a droplet digital PCR assay for the evaluation of PML-RARα fusion transcripts in acute promyelocytic leukemia

Jiang, Xiaoqian; Chen, Si-Ze; Zhu, Xia; Xu, Xiaohong; Liu, Yue

doi:10.1016/j.mcp.2020.101617

Cited by 7 publications

(4 citation statements)

References 34 publications

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“…The observation may be related to changes in the patient’s condition. Previous studies have shown that the mutation rate of promyelocytic leukemia protein-retinoic acid receptor α decreases with improving the patient’s condition in patients with acute promyelocytic leukemia; the mutation rate will increase again in relapsing patients [ 15 ]. So we considered that the difference in three ddPCR results for RP34 patient was due to the changes in patients' conditions.…”

Section: Discussionmentioning

confidence: 99%

Dynamic monitoring of UBA1 somatic mutations in patients with relapsing polychondritis

Duan,

Luo,

Wang

et al. 2024

Orphanet J Rare Dis

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Background Commonly clinically diagnosed with relapsing polychondritis (RP), vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome (VEXAS) is a recently identified autoinflammatory disease caused by UBA1 somatic mutations. The low frequency and dynamic changes challenge the accurate detection of somatic mutations. The present study monitored these mutations in Chinese patients with RP. We included 44 patients with RP. Sanger sequencing of UBA1 was performed using genomic DNA from peripheral blood. Droplet digital polymerase chain reaction (ddPCR) was performed to screen low-prevalence somatic variants. Results Multiple ddPCR detections were performed using available blood samples collected at different follow-up time points. Three male patients were UBA1 somatic mutation carriers. Sanger sequencing detected the somatic UBA1 variant c.122T > C (p.Met41Thr) in two male patients. Initial ddPCR confirmed the variant in the two patients, with allele fractions of 73.75% and 88.46%, respectively, while yielding negative results in other patients. Subsequent ddPCR detected the somatic variant (c.122T > C) with low prevalence (1.02%) in another male patient from blood samples collected at a different time point, and confirmed dynamically fractional abundance in one patient with VEXAS, with allele fractions of 73.75%, 61.28%, 65.01%, and 73.75%. Nine patients assessed by ddPCR at different time points remained negative. Conclusion We report UBA1 variants in patients with RP in the Chinese population for the first time. Multiple ddPCR detections from samples collected at different time points can enhance sensitivity and should be considered for patients with initial negative ddPCR results.

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Section: Discussionmentioning

confidence: 99%

Dynamic monitoring of UBA1 somatic mutations in patients with relapsing polychondritis

Duan,

Luo,

Wang

et al. 2024

Orphanet J Rare Dis

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“…Due to there being few reports on the FIP1L1::RARA fusion gene, studies on the pathogenesis, treatment, and prognosis of APL with FIP1L1::RARA are limited. Since it is an aggressive disease ( 12 ), there exists a high possibility of early mortality if clinicians are unable to identify an abnormal karyotype that appeared singularly or as part of a complex mutation ( 13 ). Identifying variant RARA rearrangements is critical for the diagnosis and treatment of patients with APL ( 14 ).…”

Section: Discussionmentioning

confidence: 99%

Acute promyelocytic leukemia with FIP1L1::RARA fusion gene: The clinical utility of transcriptome sequencing and bioinformatic analyses

et al. 2023

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BackgroundAcute promyelocytic leukemia (APL) is typically characterized by the presence of coagulopathy and the PML::RARA fusion gene. The FIP1L1::RARA has been reported as a novel fusion gene, but studies on its pathogenesis are limited.ObjectivesA FIP1L1::RARA fusion in a child finally diagnosed as APL was reported. RNA sequencing (RNA-seq) of six patients (three cases of acute lymphoblastic leukemia (ALL), one case of myelodysplastic syndrome (MDS), one case of acute megakaryoblastic leukemia (M7), and one case of APL with FIP1L1::RARA) were performed.MethodsTranscriptome analysis of six patients was performed by RNA-seq. The heat map was used for showing the RNA expression profile, the volcano plot for identifying differential expression genes (DEGs), and the KEGG Orthology-Based Annotation System (KOBAS) online biological information database for KEGG pathway enrichment analysis.ResultsObvious differences between APL with FIP1L1::RARA and hematologic malignancies were identified. 1060 common differentially expressed genes (co-DEGs) were detected between APL with FIP1L1::RARA vs ALL and APL with FIP1L1::RARA vs myeloid neoplasms (MDS, M7), the up-regulated genes were mainly mapped into platelet activation, cancer, AMPK signaling pathway, PI3K-Akt signaling pathway, and MAPK signaling pathway. The down-regulated genes were significantly associated with TNF signaling pathway, Rap1 signaling pathway, Age-RAGE signaling pathway, and apoptosis.ConclusionA FIP1L1::RARA fusion in a child finally diagnosed as APL was reported. RNA-seq may provide a new diagnostic method when RARA rearrangements fail to be identified by conventional methods. In the analysis of co-DEGs between case vs ALL and case vs myeloid neoplasms, the up-regulated and down-regulated genes were enriched in different signaling pathways. Further experimental studies are needed to identify pathogenesis and treatment for APL with FIP1L1::RARA.

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“…A droplet digital PCR (ddPCR) technique was utilised to directly and precisely quantify the copy numbers of nucleic acids extracted from viral and bacterial cultures. Herein, a One-Step ddPCR Advanced kit for Probes (Bio-Rad Laboratories, Hercules, CA) was used in accordance with the manufacturer’s recommendation and as previously described 36 . Briefly, the reaction mixtures were assembled with 5 µl ddPCR supermix, 40 U Reverse Transcriptase, 1 µl 300 mM Dithiothreitol (DTT), the same TaqMan primers and probes used in the multiplex real-time RT-PCR (final concentration of 500 and 250 nM, respectively), and 5 µl template nucleic acids in a final volume of 20 µl.…”

Section: Methodsmentioning

confidence: 99%

Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections

Jiang

Huang

Xie

et al. 2022

Sci Rep

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Acute respiratory infections are widespread in vulnerable populations of all ages and are characterized by a variety of symptoms. The underlying infection can be caused by a multitude of microorganisms, including viruses and bacteria. Early detection of respiratory infections through rapid pathogen screening is vital in averting infectious respiratory disease epidemics. This study utilized a multiplex real-time PCR system to develop a three-tube reverse transcription-PCR (RT-PCR) assay, enabling simultaneously detect nine respiratory pathogens, including: influenza A and B, adenovirus, respiratory syncytial virus (RSV), Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Chlamydia pneumoniae, and Mycoplasma pneumoniae. This technique utilizes a one-step assay, with specifically designed TaqMan primer–probe sets combined in the same tube. This assay provided rapid and simplified detection of the nine prevalent pathogens, as well as increased sensitivity and reduced cross-contamination. This assay was evaluated using 25 related viral/bacterial strains as positive references, the other 25 irrelevant strains as negative controls, and clinical specimens from 179 patients. All positive strains were detected with no amplification of the non-target microorganism mixtures and the assay’s detection limits ranged between 250–500 copies/ml (1.25–2.5 copies/reaction). A total of 167 (93.3%) samples tested positive for at least one of the pathogens identified; 109 of these samples were from patients confirmed to have RSV infections. The diagnostic accuracy of our assay was further confirmed by matching results from classical direct immunofluorescence assay and nucleotide sequencing. These data demonstrate the innovative multiplex real-time PCR assay as a promising alternative to the current approaches used for early screening of acute respiratory infections.

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Development and validation of a droplet digital PCR assay for the evaluation of PML-RARα fusion transcripts in acute promyelocytic leukemia

Cited by 7 publications

References 34 publications

Dynamic monitoring of UBA1 somatic mutations in patients with relapsing polychondritis

Dynamic monitoring of UBA1 somatic mutations in patients with relapsing polychondritis

Acute promyelocytic leukemia with FIP1L1::RARA fusion gene: The clinical utility of transcriptome sequencing and bioinformatic analyses

Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections

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