Validation of a Long-Read PCR Assay for Sensitive Detection and Sizing of C9orf72 Hexanucleotide Repeat Expansions

Publication date: Available online 20 August 2018Source: The Journal of Molecular DiagnosticsAuthor(s): EunRan Suh, Kaitlyn Grando, Vivianna M. Van DeerlinA hexanucleotide GGGGCC repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). Accurate determination and quantitation of the repeat length is critical in both clinical and research settings. However, due to the complexity of the C9orf72 expansion with high GC content, large size of repeats, and high rate of insertion/deletions (indels) and sequence variations in the flanking regions, molecular genetic analysis of the locus is challenging. To improve the performance characteristics for clinical testing, we evaluated a commercially available long-read C9orf72 PCR assay for research use only, AmplideX-C9, and compared its performance to our existing laboratory-developed procedure (Penn-C9). Overall, in comparison to Penn-C9, AmplideX-C9 demonstrated a more efficient workflow, greater PCR efficiency for sizing of repeat expansions, and improved peak amplitude with lower DNA input and higher analytic sensitivity. This, in turn, permitted detection of indels in the 3′ downstream of the repeat expansion region in expanded alleles, showed a higher success rate with formalin-fixed, paraffin-embedded tissue specimen, and facilitated the assessment of repeat mosaicism. In summary, AmplideX-C9 will not only help to improve clinical testing for C9orf7...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research