title: Identification of novel informative loci for DNA-based X-inactivation analysis.
abstract: The HUMARA assay, the most common method for evaluation of X-inactivation skewing in blood cells, has been reported to be usable in only about 80% of females, emphasizing the need for alternative methods for testing of HUMARA-uninformative individuals. We conducted an in silico search for potentially polymorphic tri-to-hexanucleotide repeats in the proximity of CpG islands located in 5' regions of X-chromosome genes to design five candidate assays (numbered I, II, III, IV, and V) combining methylation-specific restriction digest with PCR amplification in a manner similar to the HUMARA assay. The results obtained by these assays in 100 healthy females were compared to X-inactivation skewing measured by the AR-MSP method which is based on methylation-specific PCR amplification of the first exon of the AR gene. On the basis of statistical evidence, three of the novel assays (II, IV, and V), which were informative in 18%, 61%, and 55% of females in the cohort, respectively, may be used as alternatives or conjointly with the HUMARA assay to improve its reliability. The three new assays were combined with the HUMARA assay into a novel X-inactivation test leading to the increase of informative females in the cohort from 67% to 96%.
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic.
authors.forename: Dita
authors.initials: D
authors.lastname: Musalkova
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Minks
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Storkanova
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Dvorakova
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic. Electronic address: martin.hrebicek@lf1.cuni.cz.
authors.forename: Martin
authors.initials: M
authors.lastname: Hrebicek
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001681:Biological Assay; D016022:Case-Control Studies; D002648:Child; D041321:Chromosomes, Human, X / Q000737:chemistry*; D003198:Computer Simulation; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers / Q000138:chemical synthesis; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats; D008875:Middle Aged; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D011944:Receptors, Androgen / Q000235:genetics*; D012680:Sensitivity and Specificity; D049951:X Chromosome Inactivation
chemical_list: D017931:DNA Primers; D011944:Receptors, Androgen
keywords: Androgen receptor; Blood cells; DNA methylation; Skewing; Trinucleotide repeats; X-chromosome inactivation