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