Dataset Information

Establishment of multiplex RT-PCR to detect fusion genes for the diagnosis of Ewing sarcoma.

ABSTRACT:

Background

Detection of the tumor-specific EWSR1/FUS-ETS fusion gene is essential to diagnose Ewing sarcoma. Reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization are commonly used to detect the fusion gene, and assays using next-generation sequencing have recently been reported. However, at least 28 fusion transcript variants have been reported, making rapid and accurate detection difficult.

Methods

We constructed two sets of multiplex PCR assays and evaluated their utility using cell lines and clinical samples.

Results

EWSR1/FUS-ETS was detected in five of six tumors by the first set, and in all six tumors by the second set. The fusion gene detected only by the latter was EWSR1-ERG, which completely lacked exon 7 of EWSR1. The fusion had a short N-terminal region of EWSR1 and showed pathologically atypical features.

Conclusions

We developed multiplex RT-PCR assays to detect EWSR1-ETS and FUS-ETS simultaneously. These assays will aid the rapid and accurate diagnosis of Ewing sarcoma. In addition, variants of EWSR1/FUS-ETS with a short N-terminal region that may have been previously missed can be easily detected.

SUBMITTER: Ueno-Yokohata H

PROVIDER: S-EPMC8573982 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Establishment of multiplex RT-PCR to detect fusion genes for the diagnosis of Ewing sarcoma.

Ueno-Yokohata Hitomi H Okita Hajime H Nakasato Keiko K Kiyotani Chikako C Kato Motohiro M Matsumoto Kimikazu K Kiyokawa Nobutaka N Nakazawa Atsuko A Yoshioka Takako T

Diagnostic pathology 20211108 1

<h4>Background</h4>Detection of the tumor-specific EWSR1/FUS-ETS fusion gene is essential to diagnose Ewing sarcoma. Reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization are commonly used to detect the fusion gene, and assays using next-generation sequencing have recently been reported. However, at least 28 fusion transcript variants have been reported, making rapid and accurate detection difficult.<h4>Methods</h4>We constructed two sets of multiplex PC ...[more]

PMID: 34749732

Similar Datasets

Project description:BACKGROUND: Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. METHODS: In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. RESULTS: A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. CONCLUSION: The real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.

Dataset Information

Establishment of multiplex RT-PCR to detect fusion genes for the diagnosis of Ewing sarcoma.

Background

Methods

Results

Conclusions

Publications

Establishment of multiplex RT-PCR to detect fusion genes for the diagnosis of Ewing sarcoma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets