Abstract
BACKGROUND: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1)CpGisland 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS: Wedescribe 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, MSQMA 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, MSQMAcorrelated 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 963-973 |
| Number of pages | 11 |
| Journal | Clinical Chemistry |
| Volume | 60 |
| Issue number | 7 |
| DOIs | |
| State | Published - 2014 |
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ASJC Scopus subject areas
- Clinical Biochemistry
- Biochemistry, medical
Cite this
Early detection of fragile X syndrome : Applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots. / Inaba, Yoshimi; Schwartz, Charles E.; Bui, Quang M.; Li, Xin; Skinner, Cindy; Field, Michael; Wotton, Tiffany; Hagerman, Randi J; Francis, David; Amor, David J.; Hopper, John L.; Loesch, Danuta Z.; Bretherton, Lesley; Slater, Howard R.; Godler, David E.
In: Clinical Chemistry, Vol. 60, No. 7, 2014, p. 963-973.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Early detection of fragile X syndrome
T2 - Applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots
AU - Inaba, Yoshimi
AU - Schwartz, Charles E.
AU - Bui, Quang M.
AU - Li, Xin
AU - Skinner, Cindy
AU - Field, Michael
AU - Wotton, Tiffany
AU - Hagerman, Randi J
AU - Francis, David
AU - Amor, David J.
AU - Hopper, John L.
AU - Loesch, Danuta Z.
AU - Bretherton, Lesley
AU - Slater, Howard R.
AU - Godler, David E.
PY - 2014
Y1 - 2014
N2 - BACKGROUND: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1)CpGisland 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS: Wedescribe 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, MSQMA 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, MSQMAcorrelated 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.
AB - BACKGROUND: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1)CpGisland 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS: Wedescribe 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, MSQMA 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, MSQMAcorrelated 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=84903731689&partnerID=8YFLogxK
U2 - 10.1373/clinchem.2013.217331
DO - 10.1373/clinchem.2013.217331
M3 - Article
C2 - 24778142
AN - SCOPUS:84903731689
VL - 60
SP - 963
EP - 973
JO - Clinical Chemistry
JF - Clinical Chemistry
SN - 0009-9147
IS - 7
ER -