Early detection of fragile X syndrome: Applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots

Yoshimi Inaba, Charles E. Schwartz, Quang M. Bui, Xin Li, Cindy Skinner, Michael Field, Tiffany Wotton, Randi J Hagerman, David Francis, David J. Amor, John L. Hopper, Danuta Z. Loesch, Lesley Bretherton, Howard R. Slater, David E. Godler

Research output: Contribution to journalArticle

32 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)963-973
Number of pages11
JournalClinical Chemistry
Volume60
Issue number7
DOIs
StatePublished - 2014

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Fragile X Syndrome
Methylation
Blood
Mutation
Intellectual Disability
Alleles
Parturition
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
DNA Methylation
Southern Blotting
Routine Diagnostic Tests
Introns
Mass spectrometry
Real-Time Polymerase Chain Reaction
Assays
Mass Spectrometry
Throughput
Sensitivity and Specificity

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 journalArticle

Inaba, Y, Schwartz, CE, Bui, QM, Li, X, Skinner, C, Field, M, Wotton, T, Hagerman, RJ, Francis, D, Amor, DJ, Hopper, JL, Loesch, DZ, Bretherton, L, Slater, HR & Godler, DE 2014, 'Early detection of fragile X syndrome: Applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots', Clinical Chemistry, vol. 60, no. 7, pp. 963-973. https://doi.org/10.1373/clinchem.2013.217331
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. / Early detection of fragile X syndrome : Applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots. In: Clinical Chemistry. 2014 ; Vol. 60, No. 7. pp. 963-973.
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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.",
author = "Yoshimi Inaba and Schwartz, {Charles E.} and Bui, {Quang M.} and Xin Li and Cindy Skinner and Michael Field and Tiffany Wotton and Hagerman, {Randi J} and David Francis and Amor, {David J.} and Hopper, {John L.} and Loesch, {Danuta Z.} and Lesley Bretherton and Slater, {Howard R.} and Godler, {David E.}",
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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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