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Comparative Study
. 2004 May;6(2):96-100.
doi: 10.1016/S1525-1578(10)60496-5.

Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay

Claudia Cagnoli  1 , Chiara Michielotto, Tohru Matsuura, Tetsuo Ashizawa, Russell L Margolis, Susan E Holmes, Cinzia Gellera, Nicola Migone, Alfredo Brusco
Affiliations
Comparative Study

Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay

Claudia Cagnoli et al. J Mol Diagn. 2004 May.

Abstract

At least 18 human genetic diseases are caused by expansion of short tandem repeats. Here we describe a successful application of a fluorescent PCR method for the detection of expanded repeats in FRDA1, SCA10, and SCA12 genes. Although this test cannot give a precise estimate of the size of the expansion, it is robust, reliable, and inexpensive, and can be used to screen large series of patients. It proved useful for confirming the presence of large expansions in the Friedreich ataxia gene following an ambiguous result of long-range PCR, as well as rapid pre-screening for large repeat expansions associated with Friedreich ataxia and SCA10 and the shorter repeat expansions associated with SCA12.

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Figures

Figure 1
Figure 1
Fluorescent repeat-primed PCR analysis of the Friedreich ataxia locus. The peak profiles of healthy controls with two alleles in the normal size range (panels 1 and 2) are distinct from the profiles found in subjects with one (Hz carrier, panel 3) or two pathogenic alleles (FA patients, panels 4 and 5). The genotype of FRDA1 GAA triplets is shown inside each panel. Marker peaks of 250, 300, 340, and 350 bp are shadowed. Size in bp is also reported on abscissa, while ordinate shows an arbitrary fluorescence intensity scale. The hyphened arrow marks the right tail shape of the peak traces.
Figure 2
Figure 2
Measurement of the slope of the peaks profile. Height (F.U.) and size (bp) of repeat-primed PCR peaks can be plotted to calculated a regression line and an R2 coefficient (see Materials and Methods). A control subject and a patient corresponding to panels 2 and 5 in Figure 1 are displayed as example. The R2 values were always able to discriminate a carrier of one or two expansions versus a normal control (see Table 2).
Figure 3
Figure 3
Fluorescent repeat-primed PCR analysis in the SCA10 (left) and SCA12 (right) loci. Top panels show healthy control subjects, while bottom panels display patients carrying one expanded allele. The genotype at the two loci expressed in number of repeats is reported inside each panel. Marker peaks of 139, 150, 160, 200, 250, 300, 340, and 350 bp are shadowed. Size in bp is also reported on abscissa, while ordinate shows an arbitrary fluorescence intensity scale.
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