Yoshimi Inaba1, Charles E Schwartz2, Quang M Bui3, Xin Li1, Cindy Skinner2, Michael Field4, Tiffany Wotton5, Randi J Hagerman6, David Francis1, David J Amor7, John L Hopper3, Danuta Z Loesch8, Lesley Bretherton9, Howard R Slater7, David E Godler10. 1. Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; 2. Center for Molecular Studies, J.C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC; 3. Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Carlton, Victoria, Australia; 4. Genetics of Learning Disability Service, New South Wales, Australia; 5. New South Wales Newborn Screening Program, Children's Hospital at Westmead, Sydney, New South Wales, Australia; 6. The MIND Institute, University of California, Davis Medical Center, Sacramento, CA; Department of Pediatrics, University of California, Davis School of Medicine, Sacramento, CA; 7. Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; Department of Paediatrics, University of Melbourne, Melbourne Victoria, Australia; 8. School of Psychological Science, La Trobe University, Melbourne, Victoria, Australia.; 9. Department of Paediatrics, University of Melbourne, Melbourne Victoria, Australia; Melbourne School of Psychological Sciences, University of Melbourne; Melbourne Victoria, Australia; Department of Clinical Psychology, The Royal Children's Hospital, Melbourne; Victoria, Australia. 10. Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia; david.godler@mcri.edu.au.
Abstract
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.
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.
Authors: Kim M Cornish; Claudine M Kraan; Quang Minh Bui; Mark A Bellgrove; Sylvia A Metcalfe; Julian N Trollor; Darren R Hocking; Howard R Slater; Yoshimi Inaba; Xin Li; Alison D Archibald; Erin Turbitt; Jonathan Cohen; David E Godler Journal: Neurology Date: 2015-03-25 Impact factor: 9.910
Authors: Emma K Baker; Sheena Arora; David J Amor; Perrin Date; Meagan Cross; James O'Brien; Chloe Simons; Carolyn Rogers; Stephen Goodall; Jennie Slee; Chris Cahir; David E Godler Journal: J Autism Dev Disord Date: 2021-07-22
Authors: David E Godler; Yoshimi Inaba; Charles E Schwartz; Quang M Bui; Elva Z Shi; Xin Li; Amy S Herlihy; Cindy Skinner; Randi J Hagerman; David Francis; David J Amor; Sylvia A Metcalfe; John L Hopper; Howard R Slater Journal: Expert Rev Mol Med Date: 2015-07-01 Impact factor: 5.600