Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

Abstract

Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but also involve post-PCR processing steps. Here, we evaluated a commercially available PCR-based assay that uses melt curve analysis as a screening tool for the rapid detection of CGG repeat expansions in the fragile X mental retardation 1 (FMR1) gene. On the basis of testing with well-characterized DNA samples, the assay gave a detection limit of 10 ng per reaction and an analytic specificity beyond 150 ng per reaction. Furthermore, the melt temperatures critical for result interpretation were found to be closely linked to the CGG expansion lengths with great consistency (CV < 0.55%). The clinical performance of the assay was established with 528 blinded and previously analyzed clinical samples, yielding results of 100% sensitivity (95% CI, 91.0%-100%) and 99.6% specificity (95% CI, 98.5%-99.9%) in detecting expansions >55 CGG repeats in FMR1. This new approach eliminates post-PCR handling for all non-expanded samples, and exemplifies a truly efficient screening procedure.

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

“…Genomic DNA isolated from buccal swabs were used for preliminary screening, and all identified “expansion positives” were correctly verified by confirmatory molecular diagnostic tests. Lyons et al [153] and Lim et al [155] have validated the performance of this screening tool, and reported specificities of 95.1% (95% CI, 87.8% to 98.6%) and 99.6% (95% CI, 98.5% to 99.9%), respectively, while both studies recorded a sensitivity of 100% in detecting expansions. In a high NL DNA background, the assay was able to spot mosaicism for PM and FM down to 7.5% and 20%, respectively [155].…”

LGBTQ: The Molecular Mechanism and its Role in Elucidating Proportional for a Better Management

“…5-54) become higher CGG repeat (56-4000) and hypomethylation from normal (26-34) degraded to < 26 (5-25 CGG repeat). 21 Small CGG repeat should support LGBT law/psycho-social/MGMT has not previously been reported. Controversial are broad, LGBT problem are huge, increasing but attorney denies that gay people exist in their region (Times magazine, June 12, 2017, page 7)…”

Advanced technologies for the molecular diagnosis of fragile X syndrome

“…Coupled to either a methylation specific (MCA; (74)) or a multiplex methylation specific real time PCR (MM-RTPCR) (69), it can differentiate between different PCR amplicons as they melt at specific temperatures and it allows the quantification of the methylation status. In a more recent study, Lim GX, et al (75) validated this approach as screening tool for the identification of expanded FMR1 alleles, showing high analytic specificity and sensitivity. Since there is no need to analyze the amplified products by gel electrophoresis, this approach has the advantage of reducing the risk of contamination and may be more cost effective of those methodologies that require more post-PCR analysis and more expensive equipments/reagents such as capillary electrophoresis.…”

“…However, the TP-PCR with MCA approach has the limitation of being very laborious, not being able to distinguish between premutation and full mutation alleles and proven to be not as sensitive for the detection of full mutation females. Lastly and very importantly it does not provide CGG repeat size and FMR1 methylation status (75). However, it may represent a valid screening tool to use in a large population screening studies where the initial step flags for the presence of an expanded allele (>55 CGG repeats).…”

“…The development of novel PCR based approaches, particularly the ones based on the use of the triplet-primed PCR, of the melting curve analysis or of the fragile X-related epigenetic FREE2 FMR1 methylation, has changed this scenario. These methodologies, although can present with some of the issues discussed above, are able to flag the presence of an expanded allele, are rapid, can be cost-effective and importantly they work with small amount of DNA as the one provided by blood spots cards (28, 70), 75, 77) satisfying therefore, the requirement for a valid and effective screening tool for expanded alleles of the FMR1 gene in a large sample size.…”