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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 normalised melt curves (a, b) and the derivative melt peaks (c, d) of 107 archived patient DNA samples, followed by the GeneScan electropherograms (bottom) of selected samples. MCA profiles of samples carrying NL and expanded FMR1 alleles are clustered to the left and right of the ‘Indeterminate Zones’ (highlighted in grey), respectively.
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fig04: Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 107 archived patient DNA samples, followed by the GeneScan electropherograms (bottom) of selected samples. MCA profiles of samples carrying NL and expanded FMR1 alleles are clustered to the left and right of the ‘Indeterminate Zones’ (highlighted in grey), respectively.

Mentions: A total of 107 clinical samples, previously characterised by PCR and/or Southern analysis, were included in the blinded validation study of dTP-PCR assays. The patient cohort comprised 54 NL and 56 PM and/or FM expansion carriers, and had no samples with FMR1 alleles in the IM size range. In addition, we also included two sets of internal reference controls from CCR, NA20232 and NA20230, and NA20234 and NA20236 to define the cut-off temperature ranges for male and female samples, respectively. Normalised melt curves and melt peaks of all clinical samples with internal reference controls, followed by the GeneScan electropherograms of representative NL, PM and FM carriers are shown in Figure 4. As expected, in both males and females, two clusters of MCA profiles, one from NL samples exhibiting lower Tm compared with the respective 46-repeat reference control, and another from samples carrying expanded FMR1 alleles with Tm higher than that of the respective 54-repeat reference control, were generated. Consistent with the lack of IM samples, no MCA profiles were detected in the ‘Indeterminate Zone’. In general, we observed complete agreement between MCA and CE results for all clinical samples and achieved 100% sensitivity, 100% specificity, 100% positive predictive value and 100% negative predictive value for both dTP-PCR assays. Additionally, we verified the CGG repeat size and AGG interruption pattern of the FMR1 alleles of 17 NL males by Sanger sequencing and detected absolute concordance with dTP-PCR CE results (data not shown).Figure 4.


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 107 archived patient DNA samples, followed by the GeneScan electropherograms (bottom) of selected samples. MCA profiles of samples carrying NL and expanded FMR1 alleles are clustered to the left and right of the ‘Indeterminate Zones’ (highlighted in grey), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 107 archived patient DNA samples, followed by the GeneScan electropherograms (bottom) of selected samples. MCA profiles of samples carrying NL and expanded FMR1 alleles are clustered to the left and right of the ‘Indeterminate Zones’ (highlighted in grey), respectively.
Mentions: A total of 107 clinical samples, previously characterised by PCR and/or Southern analysis, were included in the blinded validation study of dTP-PCR assays. The patient cohort comprised 54 NL and 56 PM and/or FM expansion carriers, and had no samples with FMR1 alleles in the IM size range. In addition, we also included two sets of internal reference controls from CCR, NA20232 and NA20230, and NA20234 and NA20236 to define the cut-off temperature ranges for male and female samples, respectively. Normalised melt curves and melt peaks of all clinical samples with internal reference controls, followed by the GeneScan electropherograms of representative NL, PM and FM carriers are shown in Figure 4. As expected, in both males and females, two clusters of MCA profiles, one from NL samples exhibiting lower Tm compared with the respective 46-repeat reference control, and another from samples carrying expanded FMR1 alleles with Tm higher than that of the respective 54-repeat reference control, were generated. Consistent with the lack of IM samples, no MCA profiles were detected in the ‘Indeterminate Zone’. In general, we observed complete agreement between MCA and CE results for all clinical samples and achieved 100% sensitivity, 100% specificity, 100% positive predictive value and 100% negative predictive value for both dTP-PCR assays. Additionally, we verified the CGG repeat size and AGG interruption pattern of the FMR1 alleles of 17 NL males by Sanger sequencing and detected absolute concordance with dTP-PCR CE results (data not shown).Figure 4.

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