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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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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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