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Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

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 ?19,20 and BRCA1 c.135-1G>T ?5q and ?3). 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.

SUBMITTER: Whiley PJ

PROVIDER: S-EPMC4351044 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

Whiley Phillip J PJ de la Hoya Miguel M Thomassen Mads M Becker Alexandra A Brandão Rita R Pedersen Inge Sokilde IS Montagna Marco M Menéndez Mireia M Quiles Francisco F Gutiérrez-Enríquez Sara S De Leeneer Kim K Tenés Anna A Montalban Gemma G Tserpelis Demis D Yoshimatsu Toshio T Tirapo Carole C Raponi Michela M Caldes Trinidad T Blanco Ana A Santamariña Marta M Guidugli Lucia L de Garibay Gorka Ruiz GR Wong Ming M Tancredi Mariella M Fachal Laura L Ding Yuan Chun YC Kruse Torben T Lattimore Vanessa V Kwong Ava A Chan Tsun Leung TL Colombo Mara M De Vecchi Giovanni G Caligo Maria M Baralle Diana D Lázaro Conxi C Couch Fergus F Radice Paolo P Southey Melissa C MC Neuhausen Susan S Houdayer Claude C Fackenthal Jim J Hansen Thomas Van Overeem TV Vega Ana A Diez Orland O Blok Rien R Claes Kathleen K Wappenschmidt Barbara B Walker Logan L Spurdle Amanda B AB Brown Melissa A MA

Clinical chemistry 20131108 2

<h4>Background</h4>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.<h4>Methods</h4>We conducted a multicenter investigation to compare mRNA splicing assay protocols used by m ...[more]

PMID: 24212087

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Project description:BackgroundInaccurate, false or incomplete research publications may mislead readers including researchers and decision-makers. It is therefore important that such problems are identified and rectified promptly. This usually involves collaboration between the research institutions and academic journals involved, but these interactions can be problematic.MethodsThese recommendations were developed following discussions at World Conferences on Research Integrity in 2013 and 2017, and at a specially convened 3-day workshop in 2016 involving participants from 7 countries with expertise in publication ethics and research integrity. The recommendations aim to address issues surrounding cooperation and liaison between institutions (e.g. universities) and journals about possible and actual problems with the integrity of reported research arising before and after publication.ResultsThe main recommendations are that research institutions should: 1) develop mechanisms for assessing the integrity of reported research (if concerns are raised) that are distinct from processes to determine whether individual researchers have committed misconduct; 2) release relevant sections of reports of research integrity or misconduct investigations to all journals that have published research that was investigated; 3) take responsibility for research performed under their auspices regardless of whether the researcher still works at that institution or how long ago the work was done; 4) work with funders to ensure essential research data is retained for at least 10 years. Journals should: 1) respond to institutions about research integrity cases in a timely manner; 2) have criteria for determining whether, and what type of, information and evidence relating to the integrity of research reports should be passed on to institutions; 3) pass on research integrity concerns to institutions, regardless of whether they intend to accept the work for publication; 4) retain peer review records for at least 10 years to enable the investigation of peer review manipulation or other inappropriate behaviour by authors or reviewers.ConclusionsVarious difficulties can prevent effective cooperation between academic journals and research institutions about research integrity concerns and hinder the correction of the research record if problems are discovered. While the issues and their solutions may vary across different settings, we encourage research institutions, journals and funders to consider how they might improve future collaboration and cooperation on research integrity cases.

Project description:In this paper, we define the Association of Standardized Patient Educators (ASPE) Standards of Best Practice (SOBP) for those working with human role players who interact with learners in a wide range of experiential learning and assessment contexts. These human role players are variously described by such terms as standardized/simulated patients or simulated participants (SP or SPs). ASPE is a global organization whose mission is to share advances in SP-based pedagogy, assessment, research, and scholarship as well as support the professional development of its members. The SOBP are intended to be used in conjunction with the International Nursing Association for Clinical Simulation and Learning (INACSL) Standards of Best Practice: SimulationSM, which address broader simulation practices. We begin by providing a rationale for the creation of the ASPE SOBP, noting that with the increasing use of simulation in healthcare training, it is incumbent on ASPE to establish SOBP that ensure the growth, integrity, and safe application of SP-based educational endeavors. We then describe the three and a half year process through which these standards were developed by a consensus of international experts in the field. Key terms used throughout the document are defined. Five underlying values inform the SOBP: safety, quality, professionalism, accountability, and collaboration. Finally, we describe five domains of best practice: safe work environment; case development; SP training for role portrayal, feedback, and completion of assessment instruments; program management; and professional development. Each domain is divided into principles with accompanying key practices that provide clear and practical guidelines for achieving desired outcomes and creating simulations that are safe for all stakeholders. Failure to follow the ASPE SOBP could compromise the safety of participants and the effectiveness of a simulation session. Care has been taken to make these guidelines precise yet flexible enough to address the diversity of varying contexts of SP practice. As a living document, these SOBP will be reviewed and modified periodically under the direction of the ASPE Standards of Practice Committee as SP methodology grows and adapts to evolving simulation practices.

Dataset Information

Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

Publications

Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

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