Article open access publication

Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

Clinical Chemistry, Oxford University Press (OUP), ISSN 0009-9147

Volume 60, 2, 2014

DOI:10.1373/clinchem.2013.210658, Dimensions: pub.1024063729, PMC: PMC4351044, PMID: 24212087,



  1. (1) QIMR Berghofer Medical Research Institute, grid.1049.c
  2. (2) University of Queensland, grid.1003.2
  3. (3) Laboratorio de Oncología Molecular, Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain;
  4. (4) Odense University Hospital, grid.7143.1, Southern Denmark Region
  5. (5) University Hospital Cologne, grid.411097.a
  6. (6) University of Cologne, grid.6190.e
  7. (7) Maastricht University Medical Centre, grid.412966.e
  8. (8) Aalborg Hospital, grid.27530.33, North Denmark Region
  9. (9) Istituto Oncologico Veneto, grid.419546.b
  10. (10) Catalan Institute of Oncology, grid.418701.b
  11. (11) Hospital Universitari Vall d'Hebron, grid.411083.f
  12. (12) Ghent University Hospital, grid.410566.0
  13. (13) University of Chicago Medical Center, grid.412578.d
  14. (14) Paris Descartes University, grid.10992.33
  15. (15) Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK;
  16. (16) Fundación Pública Galega de Medicina Xenómica-SERGAS, Grupo de Medicina Xenómica-USC, CIBERER, IDIS, Santiago de Compostela, Spain;
  17. (17) University of Santiago de Compostela, grid.11794.3a
  18. (18) Mayo Clinic, grid.66875.3a
  19. (19) University of Melbourne, grid.1008.9
  20. (20) University of Pisa, grid.5395.a
  21. (21) City Of Hope National Medical Center, grid.410425.6
  22. (22) University of Otago, grid.29980.3a
  23. (23) Hong Kong Sanatorium and Hospital, grid.414329.9
  24. (24) Fondazione IRCCS Istituto Nazionale dei Tumori, grid.417893.0
  25. (25) Center for Genomic Medicine, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark;


BACKGROUND: Accurate 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. METHODS: We 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. RESULTS: PCR 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). CONCLUSIONS: We 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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Field Citation Ratio (FCR): 9.15

Relative Citation ratio (RCR): 2.01

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