title: 5meCpG epigenetic marks neighboring a primate-conserved core promoter short tandem repeat indicate X-chromosome inactivation.
abstract: X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates.
authors: Department of Genetics, School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors: Filipe Brum
authors: FB
authors: Machado
authors: Laboratory of Biotechnology, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil.
authors: Fabricio Brum
authors: FB
authors: Machado
authors: Laboratory of Biotechnology, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil.
authors: Milena Amendro
authors: MA
authors: Faria
authors: Laboratory of Biotechnology, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil.
authors: Viviane Lamim
authors: VL
authors: Lovatel
authors: Laboratory of Biotechnology, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil; Molecular Identification and Diagnostics Unit, Hospital Escola Álvaro Alvim, Campos dos Goytacazes, Rio de Janeiro, Brazil.
authors: Antonio Francisco
authors: AF
authors: Alves da Silva
authors: Laboratory of Molecular Genetics of Hemophilia, Institute of Experimental Medicine, National Academy of Medicine, Buenos Aires, Argentina.
authors: Claudia Pamela
authors: CP
authors: Radic
authors: Laboratory of Molecular Genetics of Hemophilia, Institute of Experimental Medicine, National Academy of Medicine, Buenos Aires, Argentina.
authors: Carlos Daniel
authors: CD
authors: De Brasi
authors: Laboratory of Biotechnology, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil.
authors: Álvaro Fabricio Lopes
authors: ÁF
authors: Rios
authors: Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, South Holland, the Netherlands.
authors: Susana Marina Chuva
authors: SM
authors: de Sousa Lopes
authors: Laboratory of Animal Morphology and Pathology, Center for Studies and Research in Wildlife, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil.
authors: Leonardo Serafim
authors: LS
authors: da Silveira
authors: Laboratory of Environmental Sciences, Sector of Studies of Ethology, Reintroduction and Conservation of Wild Animals, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil.
authors: Carlos Ramon
authors: CR
authors: Ruiz-Miranda
authors: Department of Genetics, School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors: Ester Silveira
authors: ES
authors: Ramos
authors: Laboratory of Biotechnology, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos do Goytacazes, Rio de Janeiro, Brazil; Molecular Identification and Diagnostics Unit, Hospital Escola Álvaro Alvim, Campos dos Goytacazes, Rio de Janeiro, Brazil.
authors: Enrique
authors: E
authors: Medina-Acosta
mesh_terms: D000818:Animals; D001483:Base Sequence; D002144:Callithrix; D002460:Cell Line; D041321:Chromosomes, Human, X / Q000235:genetics; D017124:Conserved Sequence; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005136:Eye Proteins / Q000235:genetics; D005260:Female; D019204:GTP-Binding Proteins; D005787:Gene Frequency; D006467:Hemophilia A / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences; D049951:X Chromosome Inactivation
chemical_list: D005136:Eye Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C072178:RP2 protein, human; D019204:GTP-Binding Proteins