Please use this identifier to cite or link to this item: https://doi.org/10.1016/j.jmoldx.2021.11.007
Title: High-Throughput Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis for Selective and Reliable Identification of Actionable FMR1 Genotypes
Authors: Rajan-Babu, Indhu-Shree
Phang, Gui-Ping 
Law, Hai-Yang 
Lee, Caroline G 
Chong, Samuel S 
Keywords: Science & Technology
Life Sciences & Biomedicine
Pathology
FRAGILE-X-SYNDROME
RAPID SCREENING TOOL
CGG-REPEAT
EXPANDED ALLELES
SYNDROME CARRIER
FULL MUTATION
PREMUTATION
GENE
INTERMEDIATE
EXPANSIONS
Issue Date: 1-Mar-2022
Publisher: ELSEVIER SCIENCE INC
Citation: Rajan-Babu, Indhu-Shree, Phang, Gui-Ping, Law, Hai-Yang, Lee, Caroline G, Chong, Samuel S (2022-03-01). High-Throughput Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis for Selective and Reliable Identification of Actionable FMR1 Genotypes. JOURNAL OF MOLECULAR DIAGNOSTICS 24 (3) : 241-252. ScholarBank@NUS Repository. https://doi.org/10.1016/j.jmoldx.2021.11.007
Abstract: Methylated FMR1 full-mutation expansions cause fragile X syndrome. FMR1 premutation carriers are susceptible to other late-onset conditions, and women with premutation are at risk of transmitting a fully expanded FMR1 allele to offspring. Identification of individuals with actionable FMR1 genotypes (full-mutation males and females, and premutation females at risk for primary ovarian insufficiency and/or having fragile X–affected offspring) can enable timely access to intervention services and genetic counseling. This study presents a rapid, first-tier test based on melting curve analysis of methylation-specific triplet-primed PCR amplicons (msTP-PCR MCA) for concurrent detection of FMR1 CGG-repeat expansions and their methylation status. The msTP-PCR MCA assay was optimized on 20 fragile X reference samples, and its performance was evaluated on 111 peripheral blood-derived DNA samples from patients who have undergone prior molecular testing with PCR and/or Southern blot analysis. The msTP-PCR MCA assay detected all samples with a methylated FMR1 CGG-repeat expansion, and had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 92.06%, 91.1%, and 100%, respectively. The msTP-PCR MCA assay identified premutation/full-mutation mosaicism down to 1%, detected skewed inactivation in females with FMR1 expansions, and enabled selective identification of all individuals with an actionable FMR1 genotype. The msTP-PCR MCA assay may aid in fragile X screening of at-risk populations and newborns and voluntary carrier screening of women of reproductive age.
Source Title: JOURNAL OF MOLECULAR DIAGNOSTICS
URI: https://scholarbank.nus.edu.sg/handle/10635/226776
ISSN: 15251578
19437811
DOI: 10.1016/j.jmoldx.2021.11.007
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