TY - JOUR
T1 - Comparison of mRNA splicing assay protocols across multiple laboratories
T2 - Recommendations for best practice in standardized clinical testing
AU - Whiley, Phillip J.
AU - De La Hoya, Miguel
AU - Thomassen, Mads
AU - Becker, Alexandra
AU - Brandão, Rita
AU - Pedersen, Inge Sokilde
AU - Montagna, Marco
AU - Menéndez, Mireia
AU - Quiles, Francisco
AU - Gutiérrez-Enríquez, Sara
AU - Leeneer, Kim De
AU - Tenés, Anna
AU - Montalban, Gemma
AU - Tserpelis, Demis
AU - Yoshimatsu, Toshio
AU - Tirapo, Carole
AU - Raponi, Michela
AU - Caldes, Trinidad
AU - Blanco, Ana
AU - Santamariña, Marta
AU - Guidugli, Lucia
AU - De Garibay, Gorka Ruiz
AU - Wong, Ming
AU - Tancredi, Mariella
AU - Fachal, Laura
AU - Ding, Yuan Chun
AU - Kruse, Torben
AU - Lattimore, Vanessa
AU - Kwong, Ava
AU - Chan, Tsun Leung
AU - Colombo, Mara
AU - De Vecchi, Giovanni
AU - Caligo, Maria
AU - Baralle, Diana
AU - Lázaro, Conxi
AU - Couch, Fergus
AU - Radice, Paolo
AU - Southey, Melissa C.
AU - Neuhausen, Susan
AU - Houdayer, Claude
AU - Fackenthal, Jim
AU - Van Overeem Hansen, Thomas
AU - Vega, Ana
AU - Diez, Orland
AU - Blok, Rien
AU - Claes, Kathleen
AU - Wappenschmidt, Barbara
AU - Walker, Logan
AU - Spurdle, Amanda B.
AU - Brown, Melissa A.
PY - 2014/2
Y1 - 2014/2
N2 - BACKGROUND: Accurate evaluation of unclassified sequence variants incancer predisposition genesis 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 splicingassay protocols usedbymembers 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 A5q 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.
AB - BACKGROUND: Accurate evaluation of unclassified sequence variants incancer predisposition genesis 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 splicingassay protocols usedbymembers 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 A5q 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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U2 - 10.1373/clinchem.2013.210658
DO - 10.1373/clinchem.2013.210658
M3 - Article
C2 - 24212087
AN - SCOPUS:84893496863
SN - 0009-9147
VL - 60
SP - 341
EP - 352
JO - Clinical chemistry
JF - Clinical chemistry
IS - 2
ER -