Leveraging the power of new molecular technologies in the clinical setting requires unprecedented awareness of limitations and drawbacks: experience of one diagnostic laboratory

Abstract

BackgroundAdvances in molecular technologies and in-silico variant prediction tools offer wide-ranging opportunities in diagnostic settings, yet they also present with significant limitations.ObjectiveHere, we contextualise the limitations of next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA) and in-silico prediction tools routinely used by diagnostic laboratories by reviewing specific experiences from our diagnostic laboratory.MethodsWe investigated discordant annotations and/or incorrect variant ‘callings’ in exons of 56 genes constituting our cardiomyopathy and connective tissue disorder NGS panels. Discordant variants and segmental duplications (SD) were queried using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool and the University of California Santa Cruz genome browser, respectively, to identify regions of high homology. Discrepant variant analyses by in-silico models were re-evaluated using updated file entries.ResultsWe observed a 5% error rate in MYH7 variant ‘calling’ using MLPA, which resulted from >90% homology of the MYH7 probe-binding site to MYH6. SDs were detected in TTN, PKP2 and MYLK. SDs in MYLK presented the highest risk (15.7%) of incorrect variant ‘calling’. The inaccurate ‘callings’ and discrepant in-silico predictions were resolved following detailed investigation into the source of error.ConclusionRecognising the limitations described here may help avoid incorrect diagnoses and leverage the power of new molecular technologies in diagnostic settings.

Genetic Diagnostic Testing for Inherited Cardiomyopathies

“…Although NGS-based technologies are equated with cutting-edge health care, recent studies highlight important limitations of NGS technologies and emphasize the need for continuous test development in a diagnostic setting. 41,42 In addition, it is recognized that careful evaluation of system impacts are needed before these new technologies can safely replace older validated technologies in the health care setting. 43 Because health care expenditures outpace economic growth in many countries (including Canada), there is a growing need to justify the use of scarce health care resources for funding of new and/or expanded genetic tests.…”

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

“…(38)(39)(40). More recent technologies such as Sanger and next-generation sequencing (i.e., RNA-Seq, single cell RNA-seq, NanoString) and advanced PCR methods (i.e., digital PCR) are similarly sensitive (41,42) but are relatively expensive or further require complex bioinformatical analysis (43,44). In contrast, our optimized RT-qPCR assay is designed specifically for cheap, robust, reproducible and sensitive analysis of gene expression, is available to almost any laboratory, and serves as a sensitive and specific alternative to protein expression.…”