RESUMO
Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct imprinted disorders characterized by genetic abnormalities at 15q11-q13. Early diagnosis of both syndromes provides improved treatment and accurate genetic counseling. Whole blood (WB) is the most common DNA source of many methodologies to detect PWS and AS, however, the need of WB makes a massive screening difficult in newborns due to economic and technical limitations. The aim of this study was to adapt a Methylation-sensitive High-Resolution Melting (MS-HRM) approach from dried blood spot (DBS) samples, assessing the different DNA isolation techniques and diagnostic performance. Over a 1-year period, we collected 125 DBS cards, of which 45 had already been diagnosed by MS-HRM (20 PWS, 1 AS, and 24 healthy individuals). We tested three different DBS-DNA extraction techniques assessing the DNA concentration and quality, followed by MS-HRM and statistical comparison. Each DBS-DNA extraction method was capable of accuracy in detecting all PWS and AS individuals. However, the efficiency to detect healthy individuals varied according to methodology. In our experience, DNA extracted from DBS analyzed by the MS-HRM methodology provides an accurate approach for genetic screening of imprinting related disorders in newborns, offering several benefits compared to traditional whole blood methods.
Assuntos
Síndrome de Angelman/sangue , Síndrome de Angelman/genética , Metilação de DNA/genética , Teste em Amostras de Sangue Seco , Triagem Neonatal , Desnaturação de Ácido Nucleico/genética , Síndrome de Prader-Willi/sangue , Síndrome de Prader-Willi/genética , Autoantígenos/genética , Humanos , Recém-Nascido , Projetos Piloto , Ribonuclease P/genéticaRESUMO
OBJECTIVE: To evaluate the effect of chronic moderate-intensity exercise upon the alterations of immune system cell function induced by energy restriction. METHODS: Forty male Wistar rats were randomly assigned to the following groups: sedentary animals fed ad libitum (SF, N = 10) or submitted to energy restriction (SER, N = 10, receiving 50% of the mean amount of chow consumed by SF); and trained animals fed ad libitum (TF, N = 10) or submitted to energy restriction (TER, N = 10), who exercised on a treadmill (at 60-65%VO(2max) 5 d.wk(-1) for 10 wk(-1), after 30 d under the restriction protocol. The incorporation of [2-(14)C]-thymidine by lymphocytes obtained from the spleen and mesenteric lymph nodes, plasma glucose and glutamine concentration, and cytokine production by cells cultivated in the presence of glutamine were measured in all groups, 24 h after the last exercise session. Two-way ANOVA and Tukey's posttest were employed for the statistical analysis. RESULTS: Training induced an increase in the proliferative response and in the production of gamma-interferon and interleukin-1 (P < 0.05) in cells from the spleen and lymph nodes of SER, in which these parameters were diminished when compared with SF (P < 0.05). SER spleen and lymph node cells produced more TNF (26 and 42%, respectively) and IL-2 (49 and 42%, respectively) than SF. The Th1-like diversion of the immune response observed in SER persisted after training. Partial recovery of the decreased SER plasma glutamine concentration and muscle glutamine synthase mRNA was observed. CONCLUSIONS: Training induced the recovery of the proliferative capacity of lymphocytes from SER, probably due to the partial restoration of plasma glutamine levels, but did not interfere with the diversion towards a Th1-type immune response induced by food restriction.