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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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Related in: MedlinePlus

Direct TP-PCR melt peaks (left) and GeneScan electropherograms (right) of NL/FM DNA mixtures. Presence of FM allele in the NL/FM DNA mixtures was confirmed by the identification of MCA peaks with higher Tm in the melting domains highlighted in pink.
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fig03: Direct TP-PCR melt peaks (left) and GeneScan electropherograms (right) of NL/FM DNA mixtures. Presence of FM allele in the NL/FM DNA mixtures was confirmed by the identification of MCA peaks with higher Tm in the melting domains highlighted in pink.

Mentions: The sensitivity of dTP-PCR assays in detecting low-level mosaicism for an expanded FM allele was tested using artificial DNA mixtures that contained 1, 2, 3, 4, 5, 10 and 20% of the FM allele. To generate these NL/FM DNA mixtures, we pooled the genomic DNAs of GM06890 (NL male, 30 repeats) and GM07862 (FM male, ~501–550 repeats) in different proportions with the total DNA input maintained constant at 100 ng. The dTP-PCR MCA of NL/FM DNA mixtures revealed efficient amplification of FM allele in the presence of predominant levels of NL allele, and enabled accurate detection of FM mosaicism from as low as 1% (Fig. 3, left). The low-level mosaicism for FM allele allowed visualisation of both NL and FM MCA peaks with discrete Tm in 1–10% NL/FM DNA mixtures. The availability of more FM template in the 20% NL/FM DNA mixture resulted in a much more prominent FM MCA peak that closely resembles the melt profile of a typical FM female. We noticed a slight reduction in the sensitivity of the dTP-PCR CE assay compared with MCA, with FM mosaicism detected down to 4% (Fig. 3, right).Figure 3.


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 melt peaks (left) and GeneScan electropherograms (right) of NL/FM DNA mixtures. Presence of FM allele in the NL/FM DNA mixtures was confirmed by the identification of MCA peaks with higher Tm in the melting domains highlighted in pink.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Direct TP-PCR melt peaks (left) and GeneScan electropherograms (right) of NL/FM DNA mixtures. Presence of FM allele in the NL/FM DNA mixtures was confirmed by the identification of MCA peaks with higher Tm in the melting domains highlighted in pink.
Mentions: The sensitivity of dTP-PCR assays in detecting low-level mosaicism for an expanded FM allele was tested using artificial DNA mixtures that contained 1, 2, 3, 4, 5, 10 and 20% of the FM allele. To generate these NL/FM DNA mixtures, we pooled the genomic DNAs of GM06890 (NL male, 30 repeats) and GM07862 (FM male, ~501–550 repeats) in different proportions with the total DNA input maintained constant at 100 ng. The dTP-PCR MCA of NL/FM DNA mixtures revealed efficient amplification of FM allele in the presence of predominant levels of NL allele, and enabled accurate detection of FM mosaicism from as low as 1% (Fig. 3, left). The low-level mosaicism for FM allele allowed visualisation of both NL and FM MCA peaks with discrete Tm in 1–10% NL/FM DNA mixtures. The availability of more FM template in the 20% NL/FM DNA mixture resulted in a much more prominent FM MCA peak that closely resembles the melt profile of a typical FM female. We noticed a slight reduction in the sensitivity of the dTP-PCR CE assay compared with MCA, with FM mosaicism detected down to 4% (Fig. 3, right).Figure 3.

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