期刊
CLINICAL CHEMISTRY
卷 60, 期 7, 页码 963-973出版社
OXFORD UNIV PRESS INC
DOI: 10.1373/clinchem.2013.217331
关键词
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资金
- NHMRC Project [1049299, 104299]
- Victorian Government's Operational Infrastructure Support Program
- Murdoch Childrens Research Institute
- Royal Children's Hospital Foundation
- South Carolina Department of Disabilities and Special Needs (SCDDSN)
- Roche
- Novartis
- Seaside Therapeutics
- Forest
- Curemark
- NHMRC Project
- NHMRC development grant [1017263]
- Martin & E.H. Flack Trust, Australia
- E.W. Al Thrasher Award, USA
BACKGROUND: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1) CpG island 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS: We describe methylation specific-quantitative melt analysis (MS-QMA) that targets 10 CpG sites, with 9 within FMR1 intron 1, to screen for FXS from birth in both sexes. The novel method combines the qualitative strengths of high-resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. Its performance was assessed in 312 control (CGG < 40), 143 premutation (PM) (CGG 56-170), 197 full mutation (FM) (CGG 200-2000), and 33 CGG size and methylation mosaic samples. RESULTS: In male and female newborn blood spots, MS-QMA differentiated FM from control alleles, with sensitivity, specificity, and positive and negative predictive values between 92% and 100%. In venous blood of FM females between 6 and 35 years of age, MS-QMA correlated most strongly with verbal IQ impairment (P = 0.002). In the larger cohort of males and females, MS-QMA correlated with reference methods Southern blot and MALDI-TOF mass spectrometry (P < 0.05), but was not significantly correlated with age. Unmethylated alleles in high-functioning FM and PM males determined by both reference methods were also unmethylated by MS-QMA. CONCLUSIONS: MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes. (C) 2014 American Association for Clinical Chemistry
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