title: 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.
authors.affiliation: Biofactory Pte Ltd., Singapore, Republic of Singapore.
authors.forename: Grace X Y
authors.initials: GX
authors.lastname: Lim
authors.affiliation: Biofactory Pte Ltd., Singapore, Republic of Singapore.
authors.forename: Yu Ling
authors.initials: YL
authors.lastname: Loo
authors.affiliation: Division of Human Genetics, Center for Biomedical Research, Faculty of Medicine, Diponegoro University, Semarang, Indonesia.
authors.forename: Farmaditya E P
authors.initials: FE
authors.lastname: Mundhofir
authors.affiliation: Division of Human Genetics, Center for Biomedical Research, Faculty of Medicine, Diponegoro University, Semarang, Indonesia.
authors.forename: Ferdy K
authors.initials: FK
authors.lastname: Cayami
authors.affiliation: Division of Human Genetics, Center for Biomedical Research, Faculty of Medicine, Diponegoro University, Semarang, Indonesia.
authors.forename: Sultana M H
authors.initials: SM
authors.lastname: Faradz
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.
authors.forename: Indhu-Shree
authors.initials: IS
authors.lastname: Rajan-Babu
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore; Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore, Republic of Singapore.
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Chong
authors.affiliation: Biofactory Pte Ltd., Singapore, Republic of Singapore. Electronic address: yvonne@thebiofactory.com.
authors.forename: Yvonne Y
authors.initials: YY
authors.lastname: Koh
authors.affiliation: Biofactory Pte Ltd., Singapore, Republic of Singapore.
authors.forename: Ming
authors.initials: M
authors.lastname: Guan
mesh_terms: D002460:Cell Line; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D019680:Trinucleotide Repeat Expansion
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1