Diagnostic Screening Workflow for Mutations in the @entity5345 and XXXX Genes.

@entity1 @entity5345 @entity2674 @entity14049 @entity400
OBJECTIVES: Screening for mutations in large genes is challenging in a molecular diagnostic environment. Sanger-based DNA sequencing methods are largely used; however, @entity400 ( @entity400 ) can accommodate increasing test demands and financial constraints. This study aimed to establish a simple workflow to amplify and screen all coding regions of the @entity5345 and @entity2674 ( @entity14049 ) genes by Sanger-based sequencing as well as to assess a @entity400 approach encompassing multiplex polymerase chain reaction (PCR) and pyrosequencing. METHODS: This study was conducted between July 2011 and April 2013. A total of 20 @entity1 were included in the study who had been referred to Genetic Health Services New Zealand (Northern Hub) for @entity14049 mutation screening. @entity1 were randomly divided into a @entity400 evaluation and validation cohort (n = 10 @entity1 each). Primers were designed to amplify all coding exons of @entity14049 (28 and 42 primer pairs, respectively). Primers overlying known variants were avoided to circumvent allelic drop-out. The @entity400 approach necessitated utilisation of a complementary fragment analysis assay to eliminate apparent false-positives at homopolymeric regions. Variants were filtered on the basis of their frequency and sequence depth. RESULTS: Sanger-based sequencing of PCR-amplified coding regions was successfully achieved. Sensitivity and specificity of the combined @entity400 /homopolymer protocol was determined to be 100% and 99.5%, respectively. CONCLUSION: In comparison to traditional Sanger-based sequencing, the @entity400 workflow led to a reduction in both cost and analysis time for @entity14049 screening. @entity400 analysis achieved high analytical sensitivity and specificity, but required complementary fragment analysis combined with Sanger-based sequencing confirmation in some instances.
