Early detection of fragile X syndrome: applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots.
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.
Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia;
Yoshimi
Y
Inaba
Center for Molecular Studies, J.C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC;
Charles E
CE
Schwartz
Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Carlton, Victoria, Australia;
Quang M
QM
Bui
Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia;
Xin
X
Li
Center for Molecular Studies, J.C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC;
Cindy
C
Skinner
Genetics of Learning Disability Service, New South Wales, Australia;
Michael
M
Field
New South Wales Newborn Screening Program, Children's Hospital at Westmead, Sydney, New South Wales, Australia;
Tiffany
T
Wotton
The MIND Institute, University of California, Davis Medical Center, Sacramento, CA; Department of Pediatrics, University of California, Davis School of Medicine, Sacramento, CA;
Randi J
RJ
Hagerman
Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia;
David
D
Francis
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;
David J
DJ
Amor
Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Carlton, Victoria, Australia;
John L
JL
Hopper
School of Psychological Science, La Trobe University, Melbourne, Victoria, Australia.;
Danuta Z
DZ
Loesch
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.
Lesley
L
Bretherton
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;
Howard R
HR
Slater
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.
David E
DE
Godler
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D003071:Cognition; D015331:Cohort Studies; D059788:Dried Blood Spot Testing; D042241:Early Diagnosis; D044127:Epigenesis, Genetic; D005260:Female; D005600:Fragile X Syndrome / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007438:Introns; D008297:Male; D008745:Methylation; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity