Table 6.

Methods to Characterize HTT CAG Repeats

Interpretation of CAG Repeat NumberExpected Results by Method
Conventional PCRTriplet-Primed PCR 1Expanded Repeat Analysis 2
Normal: ≤26Detected 3See footnote 1.Expansions can be detected, & repeat size can be approximated. 6
Intermediate: 27-35Detected 3, 4Expansions may be detected, but repeat size cannot be determined. 5
Pathogenic (reduced penetrance): 36-39Detected 3, 4
Pathogenic (full penetrance): ≥40Most alleles detected 3, 4Expansions detected, but repeat size cannot be determined. 5
1.

The design of a triplet-primed PCR (TP-PCR) assay may include conventional PCR primers to size normal repeats and detect expanded repeats in a single assay. The TP-PCR assay itself does not determine repeat size, even alleles in the normal range.

2.

Methods to detect and approximate the size of expanded repeats include long-range PCR sized by gel electrophoresis and Southern blotting. The upper limit of repeat size detected will vary by assay design, laboratory, sample, and/ or patient due to competition by the normal allele during amplification.

3.

Detection of an apparently homozygous repeat does not rule out the presence of an expanded CAG repeat; thus, testing by TP-PCR or expanded repeat analysis is required to detect a repeat expansion.

4.

PCR-based methods detect alleles up to about 115 CAG repeats [Potter et al 2004, Levin et al 2006]. Other methods may occasionally be useful to identify large CAG repeat tracts. The upper limit of repeat size detected will vary by assay design, laboratory, sample, and/or patient due to competition by the normal allele during amplification.

5.

Repeats at the lower end of this range may not show the characteristic stutter pattern that indicates an expanded allele.

6.

Southern blotting for the CAG repeat expansion has been described [Bean & Bayrak-Toydemir 2014].

From: Huntington Disease

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