RESUMO
Nonrandom X-chromosome inactivation (XCI), also known as skewing, has been documented in the blood cells of a significant proportion of normal aging women by the use of methylation-based assays at the polymorphic human androgen receptor locus (HUMARA). Recent data obtained with a new transcription-based XCI determination method, termed suppressive polymerase chain reaction (PCR), has shed controversy over the validity of XCI ratio results obtained with HUMARA. To resolve this disparity, we analyzed XCI in polymorphonuclear leukocytes of a large cohort of women aged 43 to 100 years with the use of HUMARA (n=100), a TaqMan single nucleotide polymorphism (SNP) assay (n=90), and the suppressive polymerase chain reaction (PCR) assay (n=67). The 3 methods yielded similar skewing incidences (42%, 38%, and 40%, respectively), and highly concordant XCI ratios. This confirms that the skewing of XCI ratio seen in blood cells of aging women is a bona fide and robust biologic phenomenon.
Assuntos
Envelhecimento/genética , Cromossomos Humanos X/genética , Neutrófilos/fisiologia , Reação em Cadeia da Polimerase/métodos , Inativação do Cromossomo X/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Metilação de DNA , Mecanismo Genético de Compensação de Dose , Feminino , Humanos , Pessoa de Meia-Idade , Modelos Genéticos , Reação em Cadeia da Polimerase/normas , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos TestesRESUMO
BACKGROUND/AIMS: The osteocalcin-related gene (ORG) is a mouse-specific member of the osteocalcin gene cluster predicted to encode a gamma-carboxyglutamic acid-rich protein. ORG mRNA has been predicted to encode nephrocalcin and shown to be expressed in the kidney where it could serve as an important crystallization inhibitor. To determine whether ORG encodes mouse nephrocalcin, we investigated its in vivo and in vitro expression. METHODS: We expressed fluorescent fusion ORG proteins in kidney cell lines and generated transgenic mice expressing enhanced green fluorescent protein under the control of the ORG promoter. RESULTS: ORG mRNA was shown to be expressed in mouse kidneys and in a variety of other tissues. Fusion constructs transfected in opossum kidney cells demonstrated integrity of the open reading frame with the presence of a protein of the expected molecular weight. However, kidneys from transgenic mice carrying the enhanced green fluorescent protein gene under the control of the ORG promoter (5.8 kb fragment) demonstrated no expression of the transgene in kidneys or other tissues. CONCLUSION: We conclude that ORG, the third gene of the mouse osteocalcin gene cluster is silent and unlikely to play a major role in mouse renal physiology.