title: Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.
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.
authors.affiliation: Genetics & Computational Biology Division, Queensland Institute of Medical Research, Brisbane, Queensland, Australia;
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authors.lastname: Diez
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authors.lastname: Claes
authors.forename: Barbara
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authors.lastname: Wappenschmidt
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mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods* / Q000592:standards*; D006801:Humans; D007753:Laboratories / Q000592:standards*; D015999:Multivariate Analysis; D017410:Practice Guidelines as Topic; D022821:RNA Splice Sites; D012326:RNA Splicing; D012680:Sensitivity and Specificity
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D022821:RNA Splice Sites