Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing-Reference-Cited by-同舟云学术

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

Published:2014-02-01 Issue:2 Volume:60 Page:341-352
ISSN:0009-9147
Container-title:Clinical Chemistry
language:en
Short-container-title:

Author:

Whiley Phillip J¹²,de la Hoya Miguel³,Thomassen Mads⁴,Becker Alexandra⁵⁶,Brandão Rita⁷,Pedersen Inge Sokilde⁸,Montagna Marco⁹,Menéndez Mireia¹⁰,Quiles Francisco¹⁰,Gutiérrez-Enríquez Sara¹¹,De Leeneer Kim¹²,Tenés Anna¹¹,Montalban Gemma¹¹,Tserpelis Demis⁷,Yoshimatsu Toshio¹³,Tirapo Carole¹⁴,Raponi Michela¹⁵,Caldes Trinidad³,Blanco Ana¹⁶,Santamariña Marta¹⁷,Guidugli Lucia¹⁸,de Garibay Gorka Ruiz³,Wong Ming¹⁹,Tancredi Mariella²⁰,Fachal Laura¹⁶,Ding Yuan Chun²¹,Kruse Torben⁴,Lattimore Vanessa²²,Kwong Ava²³,Chan Tsun Leung²³,Colombo Mara²⁴,De Vecchi Giovanni²⁴,Caligo Maria¹⁹,Baralle Diana¹⁵,Lázaro Conxi¹⁰,Couch Fergus¹⁷,Radice Paolo²⁴,Southey Melissa C¹⁸,Neuhausen Susan²¹,Houdayer Claude¹⁴,Fackenthal Jim¹³,Hansen Thomas Van Overeem²⁵,Vega Ana¹⁶,Diez Orland¹¹,Blok Rien⁷,Claes Kathleen¹²,Wappenschmidt Barbara⁵⁶,Walker Logan²²,Spurdle Amanda B¹,Brown Melissa A²

Affiliation:

1. Genetics & Computational Biology Division, Queensland Institute of Medical Research, Brisbane, Queensland, Australia

2. School of Chemistry and Molecular Biosciences, the University of Queensland, Queensland, Australia

3. Laboratorio de Oncología Molecular, Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain

4. Department of Clinical Genetics, Odense University Hospital, Odense C, Denmark

5. Center for Familial Breast and Ovarian Cancer, University Hospital of Cologne, Cologne, Germany

6. Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

7. Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands

8. Section of Molecular Diagnostics, Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark

9. Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV—IRCCS, Padua, Italy

10. Genetic Diagnosis Unit, Hereditary Cancer Program, Institut Català d'Oncologia, Barcelona, Spain

11. Oncogenetics Laboratory, Vall d'Hebron Institute of Oncology (VHIO), Vall d'Hebron Institut de Recerca, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain

12. Center for Medical Genetics, Ghent University Hospital, De Pintelaan, Ghent, Belgium

13. Department of Medicine, the University of Chicago Medical Center, Chicago, IL

14. Service de Génétique et U830, Institut Curie, et Université Paris Descartes, Sorbonne Paris Cité, Paris, France

15. Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK

16. Fundación Pública Galega de Medicina Xenómica-SERGAS, Grupo de Medicina Xenómica-USC, CIBERER, IDIS, Santiago de Compostela, Spain

17. Grupo de Medicina Xenómica—USC, University of Santiago de Compostela, CIBERER, IDIS, Santiago de Compostela, Spain

18. Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, NY

19. Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia

20. Istituto di Anatomia Patologica, Università di Pisa, Pisa, Italy

21. Department of Population Sciences, Beckman Research Institute of City of Hope, Duarte, CA

22. Department of Pathology, University of Otago, Christchurch, New Zealand

23. Division of Molecular Pathology, Department of Pathology, Hong Kong Sanatorium & Hospital, Hong Kong, China

24. Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

25. Center for Genomic Medicine, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark

Abstract

Abstract 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.

Funder

NHMRC

National Breast Cancer Foundation

Cancer Australia

National Cancer Institute

Xunta de Galicia

Publisher

Oxford University Press (OUP)

Subject

Biochemistry (medical),Clinical Biochemistry

Link

http://academic.oup.com/clinchem/article-pdf/60/2/341/32662323/clinchem0341.pdf