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

Phillip J. Whiley, Miguel De La Hoya, Mads Thomassen, Alexandra Becker, Rita Brandão, Inge Sokilde Pedersen, Marco Montagna, Mireia Menéndez, Francisco Quiles, Sara Gutiérrez-Enríquez, Kim De Leeneer, Anna Tenés, Gemma Montalban, Demis Tserpelis, Toshio Yoshimatsu, Carole Tirapo, Michela Raponi, Trinidad Caldes, Ana Blanco, Marta SantamariñaLucia Guidugli, Gorka Ruiz De Garibay, Ming Wong, Mariella Tancredi, Laura Fachal, Yuan Chun Ding, Torben Kruse, Vanessa Lattimore, Ava Kwong, Tsun Leung Chan, Mara Colombo, Giovanni De Vecchi, Maria Caligo, Diana Baralle, Conxi Lázaro, Fergus J Couch, Paolo Radice, Melissa C. Southey, Susan Neuhausen, Claude Houdayer, Jim Fackenthal, Thomas Van Overeem Hansen, Ana Vega, Orland Diez, Rien Blok, Kathleen Claes, Barbara Wappenschmidt, Logan Walker, Amanda B. Spurdle, Melissa A. Brown

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)341-352
Number of pages12
JournalClinical Chemistry
Volume60
Issue number2
DOIs
StatePublished - Feb 2014
Externally publishedYes

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Practice Guidelines
Assays
Breast Neoplasms
Messenger RNA
Testing
Polymerase Chain Reaction
Bioinformatics
Protein Isoforms
Genes
Computational Biology
Alleles

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

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; Pedersen, Inge Sokilde; Montagna, Marco; Menéndez, Mireia; Quiles, Francisco; Gutiérrez-Enríquez, Sara; Leeneer, Kim De; Tenés, Anna; Montalban, Gemma; Tserpelis, Demis; Yoshimatsu, Toshio; Tirapo, Carole; Raponi, Michela; Caldes, Trinidad; Blanco, Ana; Santamariña, Marta; Guidugli, Lucia; De Garibay, Gorka Ruiz; Wong, Ming; Tancredi, Mariella; Fachal, Laura; Ding, Yuan Chun; Kruse, Torben; Lattimore, Vanessa; Kwong, Ava; Chan, Tsun Leung; Colombo, Mara; De Vecchi, Giovanni; Caligo, Maria; Baralle, Diana; Lázaro, Conxi; Couch, Fergus J; 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.

In: Clinical Chemistry, Vol. 60, No. 2, 02.2014, p. 341-352.

Research output: Contribution to journalArticle

Whiley, PJ, De La Hoya, M, Thomassen, M, Becker, A, Brandão, R, Pedersen, IS, Montagna, M, Menéndez, M, Quiles, F, Gutiérrez-Enríquez, S, Leeneer, KD, Tenés, A, Montalban, G, Tserpelis, D, Yoshimatsu, T, Tirapo, C, Raponi, M, Caldes, T, Blanco, A, Santamariña, M, Guidugli, L, De Garibay, GR, Wong, M, Tancredi, M, Fachal, L, Ding, YC, Kruse, T, Lattimore, V, Kwong, A, Chan, TL, Colombo, M, De Vecchi, G, Caligo, M, Baralle, D, Lázaro, C, Couch, FJ, Radice, P, Southey, MC, Neuhausen, S, Houdayer, C, Fackenthal, J, Van Overeem Hansen, T, Vega, A, Diez, O, Blok, R, Claes, K, Wappenschmidt, B, Walker, L, Spurdle, AB & Brown, MA 2014, 'Comparison of mRNA splicing assay protocols across multiple laboratories: Recommendations for best practice in standardized clinical testing', Clinical Chemistry, vol. 60, no. 2, pp. 341-352. https://doi.org/10.1373/clinchem.2013.210658
Whiley, Phillip J. ; De La Hoya, Miguel ; Thomassen, Mads ; Becker, Alexandra ; Brandão, Rita ; Pedersen, Inge Sokilde ; Montagna, Marco ; Menéndez, Mireia ; Quiles, Francisco ; Gutiérrez-Enríquez, Sara ; Leeneer, Kim De ; Tenés, Anna ; Montalban, Gemma ; Tserpelis, Demis ; Yoshimatsu, Toshio ; Tirapo, Carole ; Raponi, Michela ; Caldes, Trinidad ; Blanco, Ana ; Santamariña, Marta ; Guidugli, Lucia ; De Garibay, Gorka Ruiz ; Wong, Ming ; Tancredi, Mariella ; Fachal, Laura ; Ding, Yuan Chun ; Kruse, Torben ; Lattimore, Vanessa ; Kwong, Ava ; Chan, Tsun Leung ; Colombo, Mara ; De Vecchi, Giovanni ; Caligo, Maria ; Baralle, Diana ; Lázaro, Conxi ; Couch, Fergus J ; 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. / Comparison of mRNA splicing assay protocols across multiple laboratories : Recommendations for best practice in standardized clinical testing. In: Clinical Chemistry. 2014 ; Vol. 60, No. 2. pp. 341-352.
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abstract = "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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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 J

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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