Whiley, Phillip J., de la Hoya, Miguel ORCID: 0000-0002-8113-1410, Thomassen, Mads, Becker, Alexandra, Brandao, Rita, Pedersen, Inge Sokilde ORCID: 0000-0002-9902-8040, Montagna, Marco, Menendez, Mireia, Quiles, Francisco, Gutierrez-Enriquez, Sara ORCID: 0000-0002-1711-6101, De Leeneer, Kim, Tenes, Anna, Montalban, Gemma ORCID: 0000-0002-6958-4759, Tserpelis, Demis, Yoshimatsu, Toshio ORCID: 0000-0003-2674-8159, Tirapo, Carole, Raponi, Michela, Caldes, Trinidad, Blanco, Ana, Santamarina, Marta, Guidugli, Lucia, Ruiz de Garibay, Gorka, Wong, Ming, Tancredi, Mariella, Fachal, Laura ORCID: 0000-0002-7256-9752, Ding, Yuan Chun, Kruse, Torben, Lattimore, Vanessa, Kwong, Ava ORCID: 0000-0002-6968-9489, Chan, Tsun Leung, Colombo, Mara ORCID: 0000-0001-5465-354X, De Vecchi, Giovanni, Caligo, Maria, Baralle, Diana ORCID: 0000-0003-3217-4833, Lazaro, Conxi, Couch, Fergus, Radice, Paolo ORCID: 0000-0001-6298-4111, Southey, Melissa C., Neuhausen, Susan, Houdayer, Claude, Fackenthal, Jim, Hansen, Thomas Van Overeem, Vega, Ana, Diez, Orland, Blok, Rien, Claes, Kathleen ORCID: 0000-0003-0841-7372, Wappenschmidt, Barbara, Walker, Logan ORCID: 0000-0003-0018-3719, 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. Clin. Chem., 60 (2). S. 341 - 353. WASHINGTON: AMER ASSOC CLINICAL CHEMISTRY. ISSN 1530-8561

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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.42612_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 Delta 19,20 and BRCA1 c.135-1G>T Delta 5q and Delta 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. (C) 2013 American Association for Clinical Chemistry

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Whiley, Phillip J.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
de la Hoya, MiguelUNSPECIFIEDorcid.org/0000-0002-8113-1410UNSPECIFIED
Thomassen, MadsUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Becker, AlexandraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Brandao, RitaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Pedersen, Inge SokildeUNSPECIFIEDorcid.org/0000-0002-9902-8040UNSPECIFIED
Montagna, MarcoUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Menendez, MireiaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Quiles, FranciscoUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Gutierrez-Enriquez, SaraUNSPECIFIEDorcid.org/0000-0002-1711-6101UNSPECIFIED
De Leeneer, KimUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Tenes, AnnaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Montalban, GemmaUNSPECIFIEDorcid.org/0000-0002-6958-4759UNSPECIFIED
Tserpelis, DemisUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Yoshimatsu, ToshioUNSPECIFIEDorcid.org/0000-0003-2674-8159UNSPECIFIED
Tirapo, CaroleUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Raponi, MichelaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Caldes, TrinidadUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Blanco, AnaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Santamarina, MartaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Guidugli, LuciaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Ruiz de Garibay, GorkaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wong, MingUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Tancredi, MariellaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Fachal, LauraUNSPECIFIEDorcid.org/0000-0002-7256-9752UNSPECIFIED
Ding, Yuan ChunUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kruse, TorbenUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Lattimore, VanessaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kwong, AvaUNSPECIFIEDorcid.org/0000-0002-6968-9489UNSPECIFIED
Chan, Tsun LeungUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Colombo, MaraUNSPECIFIEDorcid.org/0000-0001-5465-354XUNSPECIFIED
De Vecchi, GiovanniUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Caligo, MariaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Baralle, DianaUNSPECIFIEDorcid.org/0000-0003-3217-4833UNSPECIFIED
Lazaro, ConxiUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Couch, FergusUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Radice, PaoloUNSPECIFIEDorcid.org/0000-0001-6298-4111UNSPECIFIED
Southey, Melissa C.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Neuhausen, SusanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Houdayer, ClaudeUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Fackenthal, JimUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Hansen, Thomas Van OvereemUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Vega, AnaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Diez, OrlandUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Blok, RienUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Claes, KathleenUNSPECIFIEDorcid.org/0000-0003-0841-7372UNSPECIFIED
Wappenschmidt, BarbaraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Walker, LoganUNSPECIFIEDorcid.org/0000-0003-0018-3719UNSPECIFIED
Spurdle, Amanda B.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Brown, Melissa A.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-446161
DOI: 10.1373/clinchem.2013.210658
Journal or Publication Title: Clin. Chem.
Volume: 60
Number: 2
Page Range: S. 341 - 353
Date: 2014
Publisher: AMER ASSOC CLINICAL CHEMISTRY
Place of Publication: WASHINGTON
ISSN: 1530-8561
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
UNCLASSIFIED VARIANTS; BRCA2 VARIANTS; MOLECULAR DIAGNOSIS; PREDICTION; MUTATIONS; BREAST; GENES; EXPRESSION; GUIDELINES; CLASSIFICATIONMultiple languages
Medical Laboratory TechnologyMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/44616

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