RESUMO
Genomic imprinting is an important epigenetic phenomenon, wherein genes or gene clusters are marked by DNA methylation during gametogenesis. This plays a major role in several functions of normal cells, including cell differentiation, X chromosome inactivation, and the maintenance of chromatin structure, in mammalian development. The aim of this study was to investigate the possible differences in SNRPN gene methylation profiles in non-obese and obese individuals, and in children and adults. Our results did not reveal any statistical correlations between the DNA methylation profiles of the SNRPN gene in children or adult obese and non-obese groups. However, a comparison of the methylation levels with the chronological age revealed statistically significant differences between the means of methylation in adults and children (46.20 ± 5.88 and 39.40 ± 2.87, respectively; P < 0.001). Pearson's correlation analysis indicated a positive association between the level of DNA methylation and the chronological age (R2 = 0.326; P < 0.001). Therefore, we concluded that the methylation profile of the SNRPN promoter (in blood) is not a useful biomarker for determining the predisposition of an individual to obesity. Additionally, we have confirmed that SNRPN methylation increases with age, which raises further questions regarding the role of SNRPN expression during the aging process.
Assuntos
Peso Corporal/genética , Metilação de DNA , Estudos de Associação Genética , Proteínas Centrais de snRNP/genética , Adolescente , Adulto , Fatores Etários , Criança , Humanos , Pessoa de Meia-Idade , Obesidade/genética , Adulto Jovem , Proteínas Centrais de snRNP/químicaRESUMO
High-resolution melting (HRM) is considered an inexpensive, rapid, and attractive methodology for methylation analysis. In the application of the polymerase chain reaction (PCR) to methylation analysis, amplification efficiencies are biased towards unmethylated, rather than methylated, templates: a phenomenon known as PCR bias. To overcome PCR bias, primers that include CpG site(s) and are fully complementary to the methylated sequence have been proposed. However, genes mapped within imprinted regions usually present higher methylation levels, and an unusual PCR bias towards the methylated template can therefore arise. The manipulation of primer affinity attempts to overcome this problem. We attempted to show that mismatches at the primer's methylated binding sites increase the area between the 50 and 100% methylation plots on the melting curves, and may increase HRM accuracy for samples that have high methylation levels. Sets of primers for imprinted genes that included CpG sites at their binding sequences were designed, and were complementary to methylated or unmethylated templates. Primers fully complementary to methylated templates produced a very small area between the 50 and 100% methylation plots. When using primers that were fully complementary to the unmethylated sequence, we were able to increase the area between the 50 and 100% methylation plots. Therefore, when samples are highly methylated, such as targets in genes mapped in imprinted regions, we propose that primers should favor amplification of the rarest, unmethylated sequence. Primers may be designed to include one CpG at its binding site and be fully complementary to the unmethylated template.
Assuntos
Primers do DNA/metabolismo , Impressão Genômica/genética , Desnaturação de Ácido Nucleico/genética , Adulto , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Proteínas/genéticaRESUMO
In this qualitative study, we have analysed the communication established between mother-child and the nursing auxiliary personal in situations of injection medication in children. Data were classified according to the FORREST Theoretical Model. The non participative observation method was used with field note in twelve situations. The prevalence of blocking categories was confirmed as Disapproval, Depreciation and Tranquilizing the mother with stereotyped comments. The facilitating categories show subcategories such as recognizing feelings and presence of child clarifying and orientating mother or the responsible person. Authors concluded that there was prevalence of blocking categories when verbal communication resources were not used to interact with mother and child, without establishing a relationship of friendship and confidence and making the relation ineffective.