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 ∼175-180 CAG repeats, thus reducing the likelihood of requiring Southern blot analysis for any HD-affected sample.

Keywords:
HTT gene, Huntington disease, Triplet-primed PCR, Capillary electrophoresis
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© 2016 S. Karger AG, Basel
2016
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