ArticleClinical chemistry2014
Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.
Article in Clinical chemistry, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers, 1 of them a synthesis that pooled it.
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Who cites it
43 citing papers in PubMed, 1 synthesis or guideline pooled it, 69 citations in OpenAlex.
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- Functional characterization of splice variants in hereditary breast and ovarian cancer susceptibility genes.Familial cancer · 2025Article
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- Tracking updates in clinical databases increases efficiency for variant reanalysis.Genetics in medicine open · 2024Article
- The effect of a single SMARCA4 exon deletion on RNA splicing: Implications for variant classification.Molecular genetics & genomic medicine · 2023Article
- VIsoQLR: an interactive tool for the detection, quantification and fine-tuning of isoforms in selected genes using long-read sequencing.Human genetics · 2023Article
- Reclassification of variants of tumor suppressor genes based on Sanger RNA sequencing without NMD inhibition.Frontiers in genetics · 2023Article
- Splicing analyses for variants in MMR genes: best practice recommendations from the European Mismatch Repair Working Group.European journal of human genetics : EJHG · 2022Article
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- Classification of MSH6 Variants of Uncertain Significance Using Functional Assays.International journal of molecular sciences · 2021Review
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- Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of theCancers · 2020Article
- Criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for the Classification of Germline Sequence Variants in Risk Genes for Hereditary Breast and Ovarian Cancer.Geburtshilfe und Frauenheilkunde · 2020Article
- Mutational Screening of BRCA1/2 Genes as a Predictive Factor for Therapeutic Response in Epithelial Ovarian Cancer: A Consensus Guide from the Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH).Virchows Archiv : an international journal of pathology · 2020Review
- Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes.European journal of human genetics : EJHG · 2019Article
- Targeted RNA-seq successfully identifies normal and pathogenic splicing events in breast/ovarian cancer susceptibility and Lynch syndrome genes.International journal of cancer · 2019Article
- Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report.Journal of medical genetics · 2019Article
Corrections and comments
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Authors and funding
51 authors at 20 institutions in 12 countries.
Funding
Abstract
backgroundAccurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting.
methodsWe conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design.
resultsPCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp).
conclusionsWe provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.
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