Enhanced Detection and Sizing of the <b><i>HTT</i></b> CAG Repeat Expansion in Huntington Disease Using an Improved Triplet-Primed PCR Assay

Abstract

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.

Improved high sensitivity screen for Huntington disease using a one-step triplet-primed PCR and melting curve assay

“…In this study, we have coupled TP-PCR with MCA to develop an improved, inexpensive yet accurate one-step assay to rapidly rule in/out HD. Comparisons with the repeat-flanking PCR MCA assay showed that the TP-PCR MCA assay has a better detection sensitivity for expanded alleles, with accurate sizing of the expanded allele of up to at least ~180 CAG repeats directly from the TP-PCR MCA product, similar to what has been achieved with TP-PCR and CE directly from genomic DNA [19]. We also showed that samples carrying only normal alleles produced normalized melt curves and derivative melt peaks with reproducibly lower T m s compared to samples carrying an expanded allele.…”

“…The sequences of the TP-PCR primers have been described elsewhere [19]. Other TP-PCR MCA assays were performed on a LightCycler ® 480 Real-Time PCR System (Roche Diagnostics, Germany).…”

“…This is because TP-PCR products of expanded alleles generate a characteristic CE pattern that can be easily distinguished from the pattern from non-expanded alleles [17], which eliminates the need to perform labour-intensive Southern blot. The TP-PCR strategy has been used to successfully detect an expanded allele of >200 CAG repeats [18], and to detect and size an expanded allele of ~180 CAG repeats [19]. The American College of Medical Genetics and Genomics committee has also indicated that TP-PCR is the preferred method for genetic testing of HD [20].…”

Robust Preimplantation Genetic Testing of Huntington Disease by Combined Triplet-Primed PCR Analysis of the HTT CAG Repeat and Multi-Microsatellite Haplotyping

“…Compared to standard PCR, the triplet primed PCR (TP-PCR) method, which was first described by Warner et al . 22 , consistently detects the presence of the expanded allele regardless of its repeat length 27–30 . TP-PCR utilizes three primers – a fluorescently labeled gene-specific 5′ flanking primer, a tailed triplet-primed (TP) primer, and a tail primer.…”

“…We describe a versatile strategy for HD PGT-M involving the use of TP-PCR for robust detection of the HTT CAG repeat expansion 30 , in combination with multiplex-PCR analysis of 13 closely linked microsatellite markers to establish disease haplotype phase 32 . We also report the first clinical application of this strategy for IVF PGT-M of HD in an at-risk couple for which family members of the affected spouse were unavailable for development of a standalone linkage-based PGT-M.…”