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

Whiley, Phillip J., De La Hoya, Miguel, Thomassen, Mads, Becker, Alexandra, Brandão, Rita, Sokilde Pedersen, Inge, Montagna, Marco, Menéndez, Mireia, Quiles, Francisco, Gutiérrez-Enríquez, Sara, De Leeneer, Kim, Tenés, Anna, Montalban, Gemma, Tserpelis, Demis, Yoshimatsu, Toshio, Tirapo, Carole, Raponi, Michela, Caldes, Trinidad, Blanco, Ana, Santamariña, Marta, Guidugli, Lucia, Ruiz de Garibay, Gorka, Wong, Ming, Tancredi, Mariella, Fachal, Laura, Chun Ding, Yuan, Kruse, Torben, Lattimore, Vanessa, Kwong, Ava, Leung Chan, Tsun, Colombo, Mara, De Vecchi, Giovanni, Caligo, Maria, Baralle, Diana, Lázaro, Conxi, Couch, Fergus, Radice, Paolo, Southey, Melissa C., Neuhausen, Susan, Houdayer, Claude, Fackenthal, Jim, Van Overeem Hansen, Thomas, Vega, Ana, Diez, Orland, Blok, Rien, Claes, Kathleen, Wappenschmidt, Barbara, Walker, Logan, Spurdle, Amanda B. and Brown, Melissa A. (2014) Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing. Clinical Chemistry, 60 2: 341-352. doi:10.1373/clinchem.2013.210658

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Author Whiley, Phillip J.
De La Hoya, Miguel
Thomassen, Mads
Becker, Alexandra
Brandão, Rita
Sokilde Pedersen, Inge
Montagna, Marco
Menéndez, Mireia
Quiles, Francisco
Gutiérrez-Enríquez, Sara
De Leeneer, Kim
Tenés, Anna
Montalban, Gemma
Tserpelis, Demis
Yoshimatsu, Toshio
Tirapo, Carole
Raponi, Michela
Caldes, Trinidad
Blanco, Ana
Santamariña, Marta
Guidugli, Lucia
Ruiz de Garibay, Gorka
Wong, Ming
Tancredi, Mariella
Fachal, Laura
Chun Ding, Yuan
Kruse, Torben
Lattimore, Vanessa
Kwong, Ava
Leung Chan, Tsun
Colombo, Mara
De Vecchi, Giovanni
Caligo, Maria
Baralle, Diana
Lázaro, Conxi
Couch, Fergus
Radice, Paolo
Southey, Melissa C.
Neuhausen, Susan
Houdayer, Claude
Fackenthal, Jim
Van Overeem Hansen, Thomas
Vega, Ana
Diez, Orland
Blok, Rien
Claes, Kathleen
Wappenschmidt, Barbara
Walker, Logan
Spurdle, Amanda B.
Brown, Melissa A.
Title Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing
Journal name Clinical Chemistry   Check publisher's open access policy
ISSN 0009-9147
Publication date 2014-02
Year available 2013
Sub-type Article (original research)
DOI 10.1373/clinchem.2013.210658
Open Access Status DOI
Volume 60
Issue 2
Start page 341
End page 352
Total pages 12
Place of publication Washington, DC, United States
Publisher American Association for Clinical Chemistry
Collection year 2014
Language eng
Formatted 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.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Published November 8, 2013.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 10 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 11 times in Scopus Article | Citations
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Created: Fri, 28 Feb 2014, 12:55:59 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences