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 - De Leeneer, Kim
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 - Hansen, Thomas vO
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
AU - ENIGMA consortium
PY - 2014/2
Y1 - 2014/2
N2 - 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.
AB - 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.
KW - BRCA1 Protein
KW - BRCA2 Protein
KW - Genetic Predisposition to Disease
KW - Genetic Testing
KW - Humans
KW - Laboratories
KW - Multivariate Analysis
KW - Practice Guidelines as Topic
KW - RNA Splice Sites
KW - RNA Splicing
KW - Sensitivity and Specificity
U2 - 10.1373/clinchem.2013.210658
DO - 10.1373/clinchem.2013.210658
M3 - Journal article
C2 - 24212087
SN - 0009-9147
VL - 60
SP - 341
EP - 352
JO - Clinical Chemistry (Washington, DC)
JF - Clinical Chemistry (Washington, DC)
IS - 2
ER -