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Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis.

Rajan-Babu IS, Law HY, Yoon CS, Lee CG, Chong SS - Expert Rev Mol Med (2015)

Bottom Line: Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size.In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results.This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics,Yong Loo Lin School of Medicine,National University of Singapore,Singapore,Singapore.

ABSTRACT
Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

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Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 13 CCR reference male and 16 CCR reference female samples, followed by the GeneScan electropherograms of representative samples (bottom). Grey melt curves and peaks indicate the MCA profiles of the internal reference controls. GeneScan electropherograms of samples marked with asterisk (*) are shown in Figure 1.
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fig02: Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 13 CCR reference male and 16 CCR reference female samples, followed by the GeneScan electropherograms of representative samples (bottom). Grey melt curves and peaks indicate the MCA profiles of the internal reference controls. GeneScan electropherograms of samples marked with asterisk (*) are shown in Figure 1.

Mentions: Furthermore, to determine the ability of dTP-PCR assays in accurately categorising samples carrying FMR1 alleles of different CGG repeat sizes and AGG interruption patterns, we carried out a preliminary performance assessment study on 21 additional CCR reference DNAs, and also re-evaluated the eight samples used for initial assay optimisation (listed in Table 1). Figure 2 presents the dTP-PCR MCA data of 13 reference males and 16 reference females as normalised melt curves (a, b) and melt peaks (c, d), followed by the GeneScan electropherograms of representative samples. Using MCA we ascertained the FMR1 genotype class of each sample based on its Tm relative to reference controls that mark the lower and upper limits of the ‘Indeterminate Zone’. For the analysis of the reference males, the melt curve and peak temperatures of the IM male samples NA20232 and NA20230 marked the ‘Indeterminate Zone’ boundary, whereas the IM female samples NA20234 and NA20236 were selected to define the ‘Indeterminate Zone’ for the analysis of the reference females. As expected, all NL males carrying FMR1 alleles ranging in size from 30 to 41 CGG repeats, displayed lower Tms compared with that of NA20232 carrying a 46-repeat FMR1 allele with a 9 + 36 repeat-pattern, where ‘+’ indicates the position of AGG interruption relative to CGG repeats. In marked contrast, most PM and all FM males generated right-shifted MCA profiles that displayed higher Tms compared with that of NA20230 carrying a 54-repeat FMR1 allele with no AGG interruptions. A PM male (CD00014) generated a TP-PCR melt peak Tm in the ‘Indeterminate Zone’. CD00014 carries a sequence-verified 56-repeat allele (the second smallest PM allele size) with a 9 + 9 + 36 interruption pattern, which was also confirmed by its CE pattern. With the exception of this PM male, all NL, PM and FM reference males were unambiguously classified by MCA as nonexpanded or expanded.Figure 2.


Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis.

Rajan-Babu IS, Law HY, Yoon CS, Lee CG, Chong SS - Expert Rev Mol Med (2015)

Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 13 CCR reference male and 16 CCR reference female samples, followed by the GeneScan electropherograms of representative samples (bottom). Grey melt curves and peaks indicate the MCA profiles of the internal reference controls. GeneScan electropherograms of samples marked with asterisk (*) are shown in Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836207&req=5

fig02: Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 13 CCR reference male and 16 CCR reference female samples, followed by the GeneScan electropherograms of representative samples (bottom). Grey melt curves and peaks indicate the MCA profiles of the internal reference controls. GeneScan electropherograms of samples marked with asterisk (*) are shown in Figure 1.
Mentions: Furthermore, to determine the ability of dTP-PCR assays in accurately categorising samples carrying FMR1 alleles of different CGG repeat sizes and AGG interruption patterns, we carried out a preliminary performance assessment study on 21 additional CCR reference DNAs, and also re-evaluated the eight samples used for initial assay optimisation (listed in Table 1). Figure 2 presents the dTP-PCR MCA data of 13 reference males and 16 reference females as normalised melt curves (a, b) and melt peaks (c, d), followed by the GeneScan electropherograms of representative samples. Using MCA we ascertained the FMR1 genotype class of each sample based on its Tm relative to reference controls that mark the lower and upper limits of the ‘Indeterminate Zone’. For the analysis of the reference males, the melt curve and peak temperatures of the IM male samples NA20232 and NA20230 marked the ‘Indeterminate Zone’ boundary, whereas the IM female samples NA20234 and NA20236 were selected to define the ‘Indeterminate Zone’ for the analysis of the reference females. As expected, all NL males carrying FMR1 alleles ranging in size from 30 to 41 CGG repeats, displayed lower Tms compared with that of NA20232 carrying a 46-repeat FMR1 allele with a 9 + 36 repeat-pattern, where ‘+’ indicates the position of AGG interruption relative to CGG repeats. In marked contrast, most PM and all FM males generated right-shifted MCA profiles that displayed higher Tms compared with that of NA20230 carrying a 54-repeat FMR1 allele with no AGG interruptions. A PM male (CD00014) generated a TP-PCR melt peak Tm in the ‘Indeterminate Zone’. CD00014 carries a sequence-verified 56-repeat allele (the second smallest PM allele size) with a 9 + 9 + 36 interruption pattern, which was also confirmed by its CE pattern. With the exception of this PM male, all NL, PM and FM reference males were unambiguously classified by MCA as nonexpanded or expanded.Figure 2.

Bottom Line: Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size.In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results.This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics,Yong Loo Lin School of Medicine,National University of Singapore,Singapore,Singapore.

ABSTRACT
Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

Show MeSH
Related in: MedlinePlus