Defining the Performance Parameters of a Rapid Screening Tool for FMR1 CGG-Repeat Expansions Based on Direct Triplet-Primed PCR and Melt Curve Analysis

Abstract

Population-based screening for CGG-repeat expansions in the fragile X mental retardation 1 (FMR1) gene that cause fragile X syndrome can now be performed more cost-effectively and simply by combining direct triplet-primed PCR (dTP-PCR) with melting curve analysis (MCA). We have now performed a detailed technical validation to define the operational parameters for achieving robust and reliable performance of the FMR1 dTP-PCR MCA assay. We compared the assay's performance on 2 real-time PCR platforms and determined its analytic sensitivity and specificity. We also assessed the assay's performance on DNA isolated from different sources, the effect of differences in CGG-repeat length and AGG-interruption pattern on melt peak temperature (Tm), and the effect of common substances found in DNA solutions on Tms. The assay performed well in distinguishing normal from expansion-carrying samples. The assay had detection sensitivity down to 1 ng and an analytical specificity beyond 150 ng. In addition to peripheral blood DNA, analysis could also be performed on DNA from saliva, buccal swabs, and dried blood spots. Salt increased Tms, glycogen contamination had minimal effect, whereas AGG interruptions lowered Tms. The FMR1 dTP-PCR MCA screening assay is highly sensitive and specific, performs well using DNA from different sources, and is robust and reproducible when reagent concentrations are maintained across all tested samples.

Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

“…In a subsequent validation study [156], we characterized several key parameters that could influence the T m s and potentially result in an altered FMR1 genotypic classification of a sample. With an ability to detect expansions from as little as 1 ng input DNA, using genomic DNA isolated from a wide variety of sources including buccal swabs, saliva, peripheral blood and dried blood spots, the dTP-PCR MCA screen is readily adaptable to different real-time PCR systems and is a cost-effective method that is both robust and ideal for ruling out FMR1 expansions in a majority (>97%) of samples from the general population [70,71].…”

Rapid Molecular Screen of Spinocerebellar Ataxia Types 1, 2, and 3 by Triplet-Primed PCR and Melting Curve Analysis

“…However, on the basis of previously described assays for detection of FMR1 CGG repeats and DMPK CTG repeats, it is conceivable that the described assays should also work on different real-time PCR platforms using DNA isolated from different sources, such as saliva, buccal swabs, and dried blood spots, or under limited input DNAs of 1 to 3 ng. 34,37,41,42 In conclusion, the described strategy for rapid identification of CAG trinucleotide repeat expansions in SCA1, SCA2, and SCA3 allows for prospective screening of large numbers of samples without sacrificing assay sensitivity and compromising too much on its specificity. Such screening assays can also be useful in high-throughput referral and research laboratories to rapidly exclude SCA1, SCA2, and SCA3 expansions before further investigations.…”

“…The SCA1 and SCA2 plasmids' repeats included CAG repeats and non-CAG trinucleotides, with the 35-repeat pATXN1(CAG) 35(14 þ 1 þ 18) harboring two CAT interruptions at the 15 th and 17 th positions, and the 25-repeat pATXN2(CAG) 25(15 þ 9) harboring one CAA interruption at the 16 th position (results not shown). The 41-repeat SCA3 plasmid pATXN3(CAG) 41 contains CAA interruptions at the third and sixth positions and an AAG interruption at the fourth position, which is the default repeat structure at this locus. In addition, pATXN3(CAG) 41 also carries the C variant of single-nucleotide polymorphism rs12895357(G/C), which is located immediately downstream of the CAG repeat stretch, thus converting the first three nucleotides of the 3 0 flanking sequence from GGG to CGG.…”

A Single Common Assay for Robust and Rapid Fragile X Mental Retardation Syndrome Screening From Dried Blood Spots

“…We recently developed a rapid and cost-effective alternative for FXS screening, using a combinatory approach of TP-PCR and MCA ( Teo et al, 2012 ; Rajan-Babu et al, 2015 ), and also performed a technical validation of the TP-PCR MCA assay to identify optimum performance parameters ( Rajan-Babu et al, 2016 ). We have now demonstrated that this assay is equally accurate when applied to dried blood spot-derived DNA, which is the most readily available and stable DNA source in the context of large-scale or population-based screening, such as newborn screening.…”

“…We recently described a novel single-step screening strategy for rapid identification of FMR1 CGG repeat expansions based on melt curve analysis (MCA) of TP-PCR products ( Teo et al, 2012 ; Rajan-Babu et al, 2015 ). This strategy was validated on genotype-known DNA isolated from peripheral blood leukocytes or from cell cultures ( Rajan-Babu et al, 2016 ). To assess the accuracy of the TP-PCR MCA assay when performed on DNA isolated from dried blood spots, we conducted a double-blinded study using dried blood spots obtained from a high risk population, whose genotypes had previously been determined using a different screening method ( Winarni et al, 2012 ).…”