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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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dTP-PCR MCA profiles (left) and GeneScan electropherograms (right) of eight FMR1 genotype-known reference male and female DNA samples. Coriell IDs and CGG repeat sizes of the samples are indicated on the left and the melt peak temperatures (Tm) are indicated on the MCA profile of each sample. The −dF/dT values are shown on the y-axis and the temperatures (°C) are shown on the x-axis. Distribution pattern of AGG interruptions within the CGG repeat region are shown on the top right corner of each dTP-PCR GeneScan electropherogram, where a ‘+’ sign represents an AGG interruption. Number of CGG repeats is indicated by numbered black and grey arrows. Red arrowheads in the inset panels indicate the base-pair (bp) size, and the red peaks in the main panel are from a ROX-labelled internal size calibrator, whose bp sizes are indicated at the bottom of the electropherogram panel. rpts: total number of CGG repeats including AGG interruptions.
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fig01: dTP-PCR MCA profiles (left) and GeneScan electropherograms (right) of eight FMR1 genotype-known reference male and female DNA samples. Coriell IDs and CGG repeat sizes of the samples are indicated on the left and the melt peak temperatures (Tm) are indicated on the MCA profile of each sample. The −dF/dT values are shown on the y-axis and the temperatures (°C) are shown on the x-axis. Distribution pattern of AGG interruptions within the CGG repeat region are shown on the top right corner of each dTP-PCR GeneScan electropherogram, where a ‘+’ sign represents an AGG interruption. Number of CGG repeats is indicated by numbered black and grey arrows. Red arrowheads in the inset panels indicate the base-pair (bp) size, and the red peaks in the main panel are from a ROX-labelled internal size calibrator, whose bp sizes are indicated at the bottom of the electropherogram panel. rpts: total number of CGG repeats including AGG interruptions.

Mentions: We chose four male and four female Coriell Cell Repository (CCR) samples with NL, IM, PM and FM FMR1 alleles for the initial optimisation of dTP-PCR assay conditions (Fig. 1). Carriers and noncarriers of FMR1 expansion generated MCA profiles with distinctive melt peak temperatures (Tm); both NL (GM06890 and GM07175) and IM (NA20232 and NA20235) samples produced MCA profiles with Tms that ranged from 84.25 to 87.55°C, while expanded PM (GM06892 and GM06907) and FM (GM07862 and GM06852) samples yielded MCA profiles with a pronounced shift in their Tm to 89.25°C or above (Fig. 1, left). Moreover, the dTP-PCR MCA profiles showed good correlation with the CE estimated CGG repeat size and structure, which is well-illustrated across both male and female samples with different FMR1 genotypes (Fig. 1, right).Figure 1.


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)

dTP-PCR MCA profiles (left) and GeneScan electropherograms (right) of eight FMR1 genotype-known reference male and female DNA samples. Coriell IDs and CGG repeat sizes of the samples are indicated on the left and the melt peak temperatures (Tm) are indicated on the MCA profile of each sample. The −dF/dT values are shown on the y-axis and the temperatures (°C) are shown on the x-axis. Distribution pattern of AGG interruptions within the CGG repeat region are shown on the top right corner of each dTP-PCR GeneScan electropherogram, where a ‘+’ sign represents an AGG interruption. Number of CGG repeats is indicated by numbered black and grey arrows. Red arrowheads in the inset panels indicate the base-pair (bp) size, and the red peaks in the main panel are from a ROX-labelled internal size calibrator, whose bp sizes are indicated at the bottom of the electropherogram panel. rpts: total number of CGG repeats including AGG interruptions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: dTP-PCR MCA profiles (left) and GeneScan electropherograms (right) of eight FMR1 genotype-known reference male and female DNA samples. Coriell IDs and CGG repeat sizes of the samples are indicated on the left and the melt peak temperatures (Tm) are indicated on the MCA profile of each sample. The −dF/dT values are shown on the y-axis and the temperatures (°C) are shown on the x-axis. Distribution pattern of AGG interruptions within the CGG repeat region are shown on the top right corner of each dTP-PCR GeneScan electropherogram, where a ‘+’ sign represents an AGG interruption. Number of CGG repeats is indicated by numbered black and grey arrows. Red arrowheads in the inset panels indicate the base-pair (bp) size, and the red peaks in the main panel are from a ROX-labelled internal size calibrator, whose bp sizes are indicated at the bottom of the electropherogram panel. rpts: total number of CGG repeats including AGG interruptions.
Mentions: We chose four male and four female Coriell Cell Repository (CCR) samples with NL, IM, PM and FM FMR1 alleles for the initial optimisation of dTP-PCR assay conditions (Fig. 1). Carriers and noncarriers of FMR1 expansion generated MCA profiles with distinctive melt peak temperatures (Tm); both NL (GM06890 and GM07175) and IM (NA20232 and NA20235) samples produced MCA profiles with Tms that ranged from 84.25 to 87.55°C, while expanded PM (GM06892 and GM06907) and FM (GM07862 and GM06852) samples yielded MCA profiles with a pronounced shift in their Tm to 89.25°C or above (Fig. 1, left). Moreover, the dTP-PCR MCA profiles showed good correlation with the CE estimated CGG repeat size and structure, which is well-illustrated across both male and female samples with different FMR1 genotypes (Fig. 1, right).Figure 1.

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