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@article{2231969,
author = {Wai, Htoo A. and Constable, Matthew and Drewes, Cosima and Davies, Ian C. and Svobodová, Eliška and Dempsey, Esther and Saggar, Anand and Homfray, Tessa and Mansour, Sahar and Douzgou, Sofia and Barr, Kate and Mercer, Catherine and Hunt, David and Douglas, Andrew G. L. and Baralle, Diana},
article_location = {New York},
article_number = {7},
doi = {http://dx.doi.org/10.1002/humu.24378},
keywords = {aberrant splicing; blood RNA; RNA-seq; RT-PCR; VUS},
language = {eng},
issn = {1059-7794},
journal = {Human Mutation},
title = {Short amplicon reverse transcription-polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis},
url = {https://onlinelibrary.wiley.com/doi/10.1002/humu.24378},
volume = {43},
year = {2022}
}
TY - JOUR
ID - 2231969
AU - Wai, Htoo A. - Constable, Matthew - Drewes, Cosima - Davies, Ian C. - Svobodová, Eliška - Dempsey, Esther - Saggar, Anand - Homfray, Tessa - Mansour, Sahar - Douzgou, Sofia - Barr, Kate - Mercer, Catherine - Hunt, David - Douglas, Andrew G. L. - Baralle, Diana
PY - 2022
TI - Short amplicon reverse transcription-polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis
JF - Human Mutation
VL - 43
IS - 7
SP - 963-970
EP - 963-970
PB - Wiley
SN - 10597794
KW - aberrant splicing
KW - blood RNA
KW - RNA-seq
KW - RT-PCR
KW - VUS
UR - https://onlinelibrary.wiley.com/doi/10.1002/humu.24378
N2 - Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.
ER -
WAI, Htoo A., Matthew CONSTABLE, Cosima DREWES, Ian C. DAVIES, Eliška SVOBODOVÁ, Esther DEMPSEY, Anand SAGGAR, Tessa HOMFRAY, Sahar MANSOUR, Sofia DOUZGOU, Kate BARR, Catherine MERCER, David HUNT, Andrew G. L. DOUGLAS and Diana BARALLE. Short amplicon reverse transcription-polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis. \textit{Human Mutation}. New York: Wiley, 2022, vol.~43, No~7, p.~963-970. ISSN~1059-7794. Available from: https://dx.doi.org/10.1002/humu.24378.
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