Enhanced Detection and Sizing of the HTT CAG Repeat Expansion in Huntington Disease Using an Improved Triplet-Primed PCR Assay.

Background: Accurate determination of the CAG repeat number is crucial for diagnostic and predictive testing for Huntington disease (HD). Current PCR-based assays can accurately size up to 110 HTT CAG repeats. Objective: To develop an improved assay capable of detecting larger CAG repeat expansions. Methods: A triplet-primed PCR (TP-PCR) assay was optimized and validated on 14 HD reference DNAs, including a sample carrying a large expansion of 180 CAG repeats. Results: All 14 HD reference samples showed 100% concordance with the previously verified allele sizes. For alleles under 45 CAGs, identical repeat sizes were obtained, while alleles larger than 46 CAGs were sized to within 1 CAG. The improved TP-PCR assay successfully detected the 180 CAG repeat allele in an affected sample. Conclusion: This method extends the detection limit of large expanded alleles to at least similar to 175-180 CAG repeats, thus reducing the likelihood of requiring Southern blot analysis for any HD-affected sample. (C) 2016 S. Karger AG, Basel