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Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Godler DE, Inaba Y, Schwartz CE, Bui QM, Shi EZ, Li X, Herlihy AS, Skinner C, Hagerman RJ, Francis D, Amor DJ, Metcalfe SA, Hopper JL, Slater HR - Expert Rev Mol Med (2015)

Bottom Line: XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs).MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals.In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

View Article: PubMed Central - PubMed

Affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.

ABSTRACT
Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

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Related in: MedlinePlus

FREE2 methylation comparisons between 50 FM males and 95 FM females assessed withMS-QMA and MALDI-TOF MS MS-QMA targets seven CpG sites within FMR1intron 1 including CpG10-12 and three sites within exon 1; whereas MALDI-TOFMSCpG10-12 MOR is specific only for methylation of CpG10-12. Note: all comparisonsbetween FM males and FM females showed P < 0.001;∗∗∗ comparisons for MS-QMA mean values, with two-sample t-test used asthe data were normally distributed; ### comparison for MALDI-TOF MSbetween median values of the FM groups, with nonparametric Mann–Whitney test usedbecause of the data not being normally distributed. The bell shaped curve representsthe expected normal distribution for the FM females methylation values if theX-inactivation at the locus were random, with mean methylation ratio of 0.75, thehigher tail of distribution at 1 and the lower tail of distribution at 0.5. FM, fullmutation; FMR1, Fragile X mental retardation 1; FREE2, fragile Xrelated epigenetic element 2; MALDI-TOF MS, Matrix-assisted laserdesorption/ionisation time of flight mass spectrometry; MS-QMA, methylation specificquantitative melt analysis; MOR, methylation output ratio.
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fig03: FREE2 methylation comparisons between 50 FM males and 95 FM females assessed withMS-QMA and MALDI-TOF MS MS-QMA targets seven CpG sites within FMR1intron 1 including CpG10-12 and three sites within exon 1; whereas MALDI-TOFMSCpG10-12 MOR is specific only for methylation of CpG10-12. Note: all comparisonsbetween FM males and FM females showed P < 0.001;∗∗∗ comparisons for MS-QMA mean values, with two-sample t-test used asthe data were normally distributed; ### comparison for MALDI-TOF MSbetween median values of the FM groups, with nonparametric Mann–Whitney test usedbecause of the data not being normally distributed. The bell shaped curve representsthe expected normal distribution for the FM females methylation values if theX-inactivation at the locus were random, with mean methylation ratio of 0.75, thehigher tail of distribution at 1 and the lower tail of distribution at 0.5. FM, fullmutation; FMR1, Fragile X mental retardation 1; FREE2, fragile Xrelated epigenetic element 2; MALDI-TOF MS, Matrix-assisted laserdesorption/ionisation time of flight mass spectrometry; MS-QMA, methylation specificquantitative melt analysis; MOR, methylation output ratio.

Mentions: Here we have explored the relationship between X-inactivation and FREE2 MS-QMA MR in alarger sample than was previously analysed with the EpiTYPER system (Ref. 6). Despite the differences in assay design, there wassignificant correlation between the two assays in blood and saliva of female controls,blood of FM males and females and in saliva of 47,XXY individuals (Table 1). The distribution of the MR values in venous blood DNA forboth assays was almost identical in FM females. It showed clear skewing towards theunmethylated state from the distributions expected if the X-inactivation were random atthis locus (Fig. 3; bell shaped curve). For bothassays, methylation in FM males was significantly elevated compared with that in FMfemales. The only difference between the two assays was evident in FM males where for theEpiTYPER system the methylation ratio was skewed towards 1, whereas the FREE2 MS-QMA MRwas normally distributed in the same samples, with the top and bottom tails ofdistribution at ~1 and 0.5, respectively. Figure 3.


Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Godler DE, Inaba Y, Schwartz CE, Bui QM, Shi EZ, Li X, Herlihy AS, Skinner C, Hagerman RJ, Francis D, Amor DJ, Metcalfe SA, Hopper JL, Slater HR - Expert Rev Mol Med (2015)

FREE2 methylation comparisons between 50 FM males and 95 FM females assessed withMS-QMA and MALDI-TOF MS MS-QMA targets seven CpG sites within FMR1intron 1 including CpG10-12 and three sites within exon 1; whereas MALDI-TOFMSCpG10-12 MOR is specific only for methylation of CpG10-12. Note: all comparisonsbetween FM males and FM females showed P < 0.001;∗∗∗ comparisons for MS-QMA mean values, with two-sample t-test used asthe data were normally distributed; ### comparison for MALDI-TOF MSbetween median values of the FM groups, with nonparametric Mann–Whitney test usedbecause of the data not being normally distributed. The bell shaped curve representsthe expected normal distribution for the FM females methylation values if theX-inactivation at the locus were random, with mean methylation ratio of 0.75, thehigher tail of distribution at 1 and the lower tail of distribution at 0.5. FM, fullmutation; FMR1, Fragile X mental retardation 1; FREE2, fragile Xrelated epigenetic element 2; MALDI-TOF MS, Matrix-assisted laserdesorption/ionisation time of flight mass spectrometry; MS-QMA, methylation specificquantitative melt analysis; MOR, methylation output ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836209&req=5

fig03: FREE2 methylation comparisons between 50 FM males and 95 FM females assessed withMS-QMA and MALDI-TOF MS MS-QMA targets seven CpG sites within FMR1intron 1 including CpG10-12 and three sites within exon 1; whereas MALDI-TOFMSCpG10-12 MOR is specific only for methylation of CpG10-12. Note: all comparisonsbetween FM males and FM females showed P < 0.001;∗∗∗ comparisons for MS-QMA mean values, with two-sample t-test used asthe data were normally distributed; ### comparison for MALDI-TOF MSbetween median values of the FM groups, with nonparametric Mann–Whitney test usedbecause of the data not being normally distributed. The bell shaped curve representsthe expected normal distribution for the FM females methylation values if theX-inactivation at the locus were random, with mean methylation ratio of 0.75, thehigher tail of distribution at 1 and the lower tail of distribution at 0.5. FM, fullmutation; FMR1, Fragile X mental retardation 1; FREE2, fragile Xrelated epigenetic element 2; MALDI-TOF MS, Matrix-assisted laserdesorption/ionisation time of flight mass spectrometry; MS-QMA, methylation specificquantitative melt analysis; MOR, methylation output ratio.
Mentions: Here we have explored the relationship between X-inactivation and FREE2 MS-QMA MR in alarger sample than was previously analysed with the EpiTYPER system (Ref. 6). Despite the differences in assay design, there wassignificant correlation between the two assays in blood and saliva of female controls,blood of FM males and females and in saliva of 47,XXY individuals (Table 1). The distribution of the MR values in venous blood DNA forboth assays was almost identical in FM females. It showed clear skewing towards theunmethylated state from the distributions expected if the X-inactivation were random atthis locus (Fig. 3; bell shaped curve). For bothassays, methylation in FM males was significantly elevated compared with that in FMfemales. The only difference between the two assays was evident in FM males where for theEpiTYPER system the methylation ratio was skewed towards 1, whereas the FREE2 MS-QMA MRwas normally distributed in the same samples, with the top and bottom tails ofdistribution at ~1 and 0.5, respectively. Figure 3.

Bottom Line: XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs).MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals.In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

View Article: PubMed Central - PubMed

Affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.

ABSTRACT
Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

Show MeSH
Related in: MedlinePlus