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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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MS-QMA and SRY copy number analysis in saliva DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 13 46,XYcontrol males (<40 CGGs); 27 46,XX control females (<40 CGGs); 3647,XXY Klinefelter syndrome males (confirmed by microarray analysis); 12 47,XXYtreated as Klinefelter syndrome males (confirmed by androgen receptor methylationanalysis and androgen receptor CAG repeat length heterozygocity; no karyotypeavailable); 5 mosaics for 46,XY/47,XXY (confirmed by microarray analysis); and 446,XX males with an SRY translocation (confirmed by microarray analysis), 2 PM/FMsize mosaic males, 1 FM male (a) The MR values were determined using MS-QMA; (b) TheSRY/β-globin copy number ratios were determined using real-time PCR relativestandard curve method. Note: the red broken line represents the maximum value of thefemale control group; the black broken line represents the optimal threshold valuefor detection of presence of one or more copies of Y chromosome. FM, full mutation;MR, methylation ratio; MS-QMA, methylation specific quantitative melt analysis; PCR,polymerase chain reaction; PM/FM, permutation/full mutation; SRY, sex determiningregion Y.
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fig01: MS-QMA and SRY copy number analysis in saliva DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 13 46,XYcontrol males (<40 CGGs); 27 46,XX control females (<40 CGGs); 3647,XXY Klinefelter syndrome males (confirmed by microarray analysis); 12 47,XXYtreated as Klinefelter syndrome males (confirmed by androgen receptor methylationanalysis and androgen receptor CAG repeat length heterozygocity; no karyotypeavailable); 5 mosaics for 46,XY/47,XXY (confirmed by microarray analysis); and 446,XX males with an SRY translocation (confirmed by microarray analysis), 2 PM/FMsize mosaic males, 1 FM male (a) The MR values were determined using MS-QMA; (b) TheSRY/β-globin copy number ratios were determined using real-time PCR relativestandard curve method. Note: the red broken line represents the maximum value of thefemale control group; the black broken line represents the optimal threshold valuefor detection of presence of one or more copies of Y chromosome. FM, full mutation;MR, methylation ratio; MS-QMA, methylation specific quantitative melt analysis; PCR,polymerase chain reaction; PM/FM, permutation/full mutation; SRY, sex determiningregion Y.

Mentions: In our earlier study using venous blood samples we have shown that the MS-QMA thresholdrange between 0.39 and 0.41 MR provided sensitivity and specificity approaching 100% fordetection of FM females with verbal IQ (VIQ) impairment (<70) (Ref. 17). The threshold of 0.37 MR was the maximum valueof the female healthy control range as well as the optimal for detection of FM femaleswith performance IQ (PIQ) and full scale IQ (FSIQ) impairment (<70). Consistentwith this, both blood and saliva DNA from female controls showed MR below the 0.37 maximumcontrol value. Three out of 57 XXY saliva samples (5%) showed MS-QMA MR above the 0.37 MRthreshold. Being above the methylation maximum in control females, representing expectedrange values for ‘normal pattern’ of X-inactivation for 2 X chromosomes in one cell, itsuggests that these 5% of XXY samples have extremely skewed X-inactivation in saliva, orpresence of cells with a third X chromosome not detected by microarray technology (Fig. 1a). It is also of interest that the saliva DNAof the FXS affected males had MS-QMA MR values well above these thresholds, whereas forthe PM/FM mosaic group one male showed an MR of 0.37. Figure 1.


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 saliva DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 13 46,XYcontrol males (<40 CGGs); 27 46,XX control females (<40 CGGs); 3647,XXY Klinefelter syndrome males (confirmed by microarray analysis); 12 47,XXYtreated as Klinefelter syndrome males (confirmed by androgen receptor methylationanalysis and androgen receptor CAG repeat length heterozygocity; no karyotypeavailable); 5 mosaics for 46,XY/47,XXY (confirmed by microarray analysis); and 446,XX males with an SRY translocation (confirmed by microarray analysis), 2 PM/FMsize mosaic males, 1 FM male (a) The MR values were determined using MS-QMA; (b) TheSRY/β-globin copy number ratios were determined using real-time PCR relativestandard curve method. Note: the red broken line represents the maximum value of thefemale control group; the black broken line represents the optimal threshold valuefor detection of presence of one or more copies of Y chromosome. FM, full mutation;MR, methylation ratio; MS-QMA, methylation specific quantitative melt analysis; PCR,polymerase chain reaction; PM/FM, permutation/full mutation; SRY, sex determiningregion Y.
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Related In: Results  -  Collection

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

fig01: MS-QMA and SRY copy number analysis in saliva DNA samples from control males andfemales and individuals with a sex chromosome aneuploidy. There were 13 46,XYcontrol males (<40 CGGs); 27 46,XX control females (<40 CGGs); 3647,XXY Klinefelter syndrome males (confirmed by microarray analysis); 12 47,XXYtreated as Klinefelter syndrome males (confirmed by androgen receptor methylationanalysis and androgen receptor CAG repeat length heterozygocity; no karyotypeavailable); 5 mosaics for 46,XY/47,XXY (confirmed by microarray analysis); and 446,XX males with an SRY translocation (confirmed by microarray analysis), 2 PM/FMsize mosaic males, 1 FM male (a) The MR values were determined using MS-QMA; (b) TheSRY/β-globin copy number ratios were determined using real-time PCR relativestandard curve method. Note: the red broken line represents the maximum value of thefemale control group; the black broken line represents the optimal threshold valuefor detection of presence of one or more copies of Y chromosome. FM, full mutation;MR, methylation ratio; MS-QMA, methylation specific quantitative melt analysis; PCR,polymerase chain reaction; PM/FM, permutation/full mutation; SRY, sex determiningregion Y.
Mentions: In our earlier study using venous blood samples we have shown that the MS-QMA thresholdrange between 0.39 and 0.41 MR provided sensitivity and specificity approaching 100% fordetection of FM females with verbal IQ (VIQ) impairment (<70) (Ref. 17). The threshold of 0.37 MR was the maximum valueof the female healthy control range as well as the optimal for detection of FM femaleswith performance IQ (PIQ) and full scale IQ (FSIQ) impairment (<70). Consistentwith this, both blood and saliva DNA from female controls showed MR below the 0.37 maximumcontrol value. Three out of 57 XXY saliva samples (5%) showed MS-QMA MR above the 0.37 MRthreshold. Being above the methylation maximum in control females, representing expectedrange values for ‘normal pattern’ of X-inactivation for 2 X chromosomes in one cell, itsuggests that these 5% of XXY samples have extremely skewed X-inactivation in saliva, orpresence of cells with a third X chromosome not detected by microarray technology (Fig. 1a). It is also of interest that the saliva DNAof the FXS affected males had MS-QMA MR values well above these thresholds, whereas forthe PM/FM mosaic group one male showed an MR of 0.37. Figure 1.

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