title: Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.
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.
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: David E
authors.initials: DE
authors.lastname: Godler
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Inaba
authors.affiliation: Center for Molecular Studies,J.C. Self Research Institute of Human Genetics,Greenwood Genetic Center,South CA,USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Schwartz
authors.affiliation: Centre for Molecular,Environmental,Genetic and Analytic Epidemiology,University of Melbourne,Carlton,Victoria,3053,Australia.
authors.forename: Quang M
authors.initials: QM
authors.lastname: Bui
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: Elva Z
authors.initials: EZ
authors.lastname: Shi
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Public Health Genetics,Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Herlihy
authors.affiliation: Center for Molecular Studies,J.C. Self Research Institute of Human Genetics,Greenwood Genetic Center,South CA,USA.
authors.forename: Cindy
authors.initials: C
authors.lastname: Skinner
authors.affiliation: The MIND Institute,University of California,Davis Medical Center,Sacramento,CA,USA.
authors.forename: Randi J
authors.initials: RJ
authors.lastname: Hagerman
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: David
authors.initials: D
authors.lastname: Francis
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Amor
authors.affiliation: Department of Paediatrics,University of Melbourne,Melbourne Victoria,3052,Australia.
authors.forename: Sylvia A
authors.initials: SA
authors.lastname: Metcalfe
authors.affiliation: Centre for Molecular,Environmental,Genetic and Analytic Epidemiology,University of Melbourne,Carlton,Victoria,3053,Australia.
authors.forename: John L
authors.initials: JL
authors.lastname: Hopper
authors.affiliation: Cyto-molecular Diagnostic Research Laboratory,Victorian Clinical Genetics Services and Murdoch Children's Research Institute,Royal Children's Hospital,Melbourne,Victoria,3052,Australia.
authors.forename: Howard R
authors.initials: HR
authors.lastname: Slater
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000782:Aneuploidy; D002648:Child; D002675:Child, Preschool; D041321:Chromosomes, Human, X; D018899:CpG Islands; D004247:DNA / Q000097:blood / Q000235:genetics*; D019175:DNA Methylation; D005091:Exons; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000097:blood / Q000235:genetics*; D005600:Fragile X Syndrome / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007438:Introns; D008297:Male; D008875:Middle Aged; D009691:Nucleic Acid Denaturation; D012463:Saliva / Q000737:chemistry; D049951:X Chromosome Inactivation
chemical_list: D004247:DNA; D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human