title: 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.
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Indhu-Shree
authors.initials: IS
authors.lastname: Rajan-Babu
authors.affiliation: Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore.
authors.forename: Mulias
authors.initials: M
authors.lastname: Lian
authors.affiliation: Department of Physiology, Vietnam Military Medical University, Hanoi, Vietnam.
authors.forename: Anh H
authors.initials: AH
authors.lastname: Tran
authors.affiliation: Department of Anatomy, Vietnam Military Medical University, Hanoi, Vietnam; Department of Genetics and Cytogenetics, Vietnam Military Medical University, Hanoi, Vietnam.
authors.forename: Truong T
authors.initials: TT
authors.lastname: Dang
authors.affiliation: National Hospital of Pediatrics, Hanoi, Vietnam.
authors.forename: Huong T-M
authors.initials: HT
authors.lastname: Le
authors.affiliation: National Hospital of Pediatrics, Hanoi, Vietnam.
authors.forename: Minh N
authors.initials: MN
authors.lastname: Thanh
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Division of Medical Sciences, National Cancer Center, Singapore, Singapore; Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, Singapore.
authors.forename: Caroline G
authors.initials: CG
authors.lastname: Lee
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore; Department of Laboratory Medicine, National University Hospital, National University Health System, Singapore, Singapore. Electronic address: paecs@nus.edu.sg.
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Chong
mesh_terms: D002460:Cell Line; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D019680:Trinucleotide Repeat Expansion; D018911:Trinucleotide Repeats
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1