Limits...
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.

Show MeSH

Related in: MedlinePlus

MS-QMA and SRY copy number analysis in blood DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 19 46,XYcontrol males (<40 CGGs); 48 46,XX control females (<40 CGGs); 447,XXY Klinefelter syndrome males; 10 45,X Turners syndrome females; 8 47,XXXfemales; 4 47,XYY males; 1 48,XXYY/47,XXY mosaic male; 2 48,XXXY males; and 449,XXXXY males. All were confirmed by microarray analysis. (a) The MR valuesdetermined using MS-QMA; (b) The SRY / β-globin copy number ratiosdetermined using real-time PCR relative standard curve method. Note: the red brokenline represent the maximum value of the female control group; the black broken lineis the threshold value that optimally separates samples with one or more copies ofthe Y chromosome. Red arrow highlights a 45,X sample with SRY/β-globin ratio abovethe positive threshold of 0.1. Because the Y chromosome was not detected usingmicroarray analysis in this sample and SRY FISH could not be performed because ofblood sample unavailability, this result may be either a false positive or a truepositive where real-time PCR may be a more sensitive approach for detection of lowlevel mosaicism. For comparisons with 46,XY controls ###P < 0.001 for SRY cioy munber and 46,XX controls ***P < 0.001 for MS-QMA MR, nonparametric Mann–Whitney testfor median was used. FISH, fluorescence in situ hybridisation; MR, methylationratio; MS-QMA, methylation specific quantitative melt analysis; PCR, polymerasechain reaction; SRY, sex determining region Y.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836209&req=5

fig02: MS-QMA and SRY copy number analysis in blood DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 19 46,XYcontrol males (<40 CGGs); 48 46,XX control females (<40 CGGs); 447,XXY Klinefelter syndrome males; 10 45,X Turners syndrome females; 8 47,XXXfemales; 4 47,XYY males; 1 48,XXYY/47,XXY mosaic male; 2 48,XXXY males; and 449,XXXXY males. All were confirmed by microarray analysis. (a) The MR valuesdetermined using MS-QMA; (b) The SRY / β-globin copy number ratiosdetermined using real-time PCR relative standard curve method. Note: the red brokenline represent the maximum value of the female control group; the black broken lineis the threshold value that optimally separates samples with one or more copies ofthe Y chromosome. Red arrow highlights a 45,X sample with SRY/β-globin ratio abovethe positive threshold of 0.1. Because the Y chromosome was not detected usingmicroarray analysis in this sample and SRY FISH could not be performed because ofblood sample unavailability, this result may be either a false positive or a truepositive where real-time PCR may be a more sensitive approach for detection of lowlevel mosaicism. For comparisons with 46,XY controls ###P < 0.001 for SRY cioy munber and 46,XX controls ***P < 0.001 for MS-QMA MR, nonparametric Mann–Whitney testfor median was used. FISH, fluorescence in situ hybridisation; MR, methylationratio; MS-QMA, methylation specific quantitative melt analysis; PCR, polymerasechain reaction; SRY, sex determining region Y.

Mentions: For venous blood DNA, only samples with three or more copies of X chromosome showedMS-QMA MR above the 0.37 threshold (Fig. 2a). Aswith our previous FREE2 methylation analyses using the EpiTYPER system (Ref. 8), the 47,XXX and 49,XXXXY groups showed MS-QMA MRsignificantly above those of female controls. As expected, saliva and venous blood DNAsamples with a Y chromosome according to previous laboratory testing also showed ~1 copyof SRY, whereas samples with two or more copies of a Y chromosome showed SRY copy numberbetween 1.5 and 2 (Figs 1b and 2b). The only exception was one 45,X sample thatconsistently showed SRY/β-globin ratio above the positive threshold of 0.1 (Fig. 2b). Because the Y chromosome was not detectedusing microarray analysis in this sample and SRY fluorescence in situ hybridisation (FISH)could not be performed because of blood sample unavailability, this result may be either afalse positive or a true positive where real-time PCR may be a more sensitive approach fordetection of low level mosaicism. Figure 2.


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)

MS-QMA and SRY copy number analysis in blood DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 19 46,XYcontrol males (<40 CGGs); 48 46,XX control females (<40 CGGs); 447,XXY Klinefelter syndrome males; 10 45,X Turners syndrome females; 8 47,XXXfemales; 4 47,XYY males; 1 48,XXYY/47,XXY mosaic male; 2 48,XXXY males; and 449,XXXXY males. All were confirmed by microarray analysis. (a) The MR valuesdetermined using MS-QMA; (b) The SRY / β-globin copy number ratiosdetermined using real-time PCR relative standard curve method. Note: the red brokenline represent the maximum value of the female control group; the black broken lineis the threshold value that optimally separates samples with one or more copies ofthe Y chromosome. Red arrow highlights a 45,X sample with SRY/β-globin ratio abovethe positive threshold of 0.1. Because the Y chromosome was not detected usingmicroarray analysis in this sample and SRY FISH could not be performed because ofblood sample unavailability, this result may be either a false positive or a truepositive where real-time PCR may be a more sensitive approach for detection of lowlevel mosaicism. For comparisons with 46,XY controls ###P < 0.001 for SRY cioy munber and 46,XX controls ***P < 0.001 for MS-QMA MR, nonparametric Mann–Whitney testfor median was used. FISH, fluorescence in situ hybridisation; MR, methylationratio; MS-QMA, methylation specific quantitative melt analysis; PCR, polymerasechain reaction; SRY, sex determining region Y.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: MS-QMA and SRY copy number analysis in blood DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 19 46,XYcontrol males (<40 CGGs); 48 46,XX control females (<40 CGGs); 447,XXY Klinefelter syndrome males; 10 45,X Turners syndrome females; 8 47,XXXfemales; 4 47,XYY males; 1 48,XXYY/47,XXY mosaic male; 2 48,XXXY males; and 449,XXXXY males. All were confirmed by microarray analysis. (a) The MR valuesdetermined using MS-QMA; (b) The SRY / β-globin copy number ratiosdetermined using real-time PCR relative standard curve method. Note: the red brokenline represent the maximum value of the female control group; the black broken lineis the threshold value that optimally separates samples with one or more copies ofthe Y chromosome. Red arrow highlights a 45,X sample with SRY/β-globin ratio abovethe positive threshold of 0.1. Because the Y chromosome was not detected usingmicroarray analysis in this sample and SRY FISH could not be performed because ofblood sample unavailability, this result may be either a false positive or a truepositive where real-time PCR may be a more sensitive approach for detection of lowlevel mosaicism. For comparisons with 46,XY controls ###P < 0.001 for SRY cioy munber and 46,XX controls ***P < 0.001 for MS-QMA MR, nonparametric Mann–Whitney testfor median was used. FISH, fluorescence in situ hybridisation; MR, methylationratio; MS-QMA, methylation specific quantitative melt analysis; PCR, polymerasechain reaction; SRY, sex determining region Y.
Mentions: For venous blood DNA, only samples with three or more copies of X chromosome showedMS-QMA MR above the 0.37 threshold (Fig. 2a). Aswith our previous FREE2 methylation analyses using the EpiTYPER system (Ref. 8), the 47,XXX and 49,XXXXY groups showed MS-QMA MRsignificantly above those of female controls. As expected, saliva and venous blood DNAsamples with a Y chromosome according to previous laboratory testing also showed ~1 copyof SRY, whereas samples with two or more copies of a Y chromosome showed SRY copy numberbetween 1.5 and 2 (Figs 1b and 2b). The only exception was one 45,X sample thatconsistently showed SRY/β-globin ratio above the positive threshold of 0.1 (Fig. 2b). Because the Y chromosome was not detectedusing microarray analysis in this sample and SRY fluorescence in situ hybridisation (FISH)could not be performed because of blood sample unavailability, this result may be either afalse positive or a true positive where real-time PCR may be a more sensitive approach fordetection of low level mosaicism. Figure 2.

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