Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.FAPESP (#2008/07370-0

Consanguinity and founder effect for Gaucher disease mutation G377S in a population from Tabuleiro do Norte, Northeastern Brazil

Gaucher’s disease (GD) is caused by a β-glucocerebrosidase deficiency,\ud leading to the accumulation of glucocerebroside in the reticuloendothelial\ud system. The prevalence of GD in Tabuleiro do Norte (TN) (1:4000) is the\ud highest in Brazil. The purpose of this study was to present evidence of\ud consanguinity and founder effect for the G377S mutation (c.1246G>A)\ud among GD patients in TN based on enzyme, molecular and genealogical\ud studies. Between March 2009 and December 2010, 131 subjects at risk for\ud GD (GC in dried blood ≤2.19 nmol/h/ml) and 5 confirmed GD patients from\ud the same community were submitted for molecular analysis to characterize\ud the genetic profile of the population. Based on the enzymatic and molecular\ud analysis, the subjects were classified into three categories: affected (n=5),\ud carrier (n=20) and non-carrier (n=111). All carriers were (G377S/wt).\ud Affected subjects were homozygous (G377S/G377S). The identification of a\ud single mutation in carriers and homozygotes from different generations, the\ud history of the community and the genealogy study suggest that the high\ud prevalence of GD in this population may be due to a combination of\ud consanguinity and founder effect for the G377S mutation.The authors would like to thank Genzyme do Brasil for financial support. Also thanks to Dr Elisa Sobreira for academic support and to Mr João Márcio da Silva (head of the Municipal Health Department) and the health care workers of the local facility for their valuable cooperation. We are grateful to Dr Rômulo Maurício for preparing blood samples for enzyme analyses and to Tibelle Freitas Maurício (nurse) for her contribution to the health education sessions. Finally, our thanks to Edineide Chaves (social worker) and to Prof Marcondes Andrade for helping draw the family trees of the involved families

Induced Pluripotent Stem Cell for the Study and Treatment of Sickle Cell Anemia

Sickle cell anemia (SCA) is a monogenic disease of high mortality, affecting millions of people worldwide. There is no broad, effective, and safe definitive treatment for SCA, so the palliative treatments are the most used. The establishment of an in vitro model allows better understanding of how the disease occurs, besides allowing the development of more effective tests and treatments. In this context, iPSC technology is a powerful tool for basic research and disease modeling, and a promise for finding and screening more effective and safe drugs, besides the possibility of use in regenerative medicine. This work obtained a model for study and treatment of SCA using iPSC. Then, episomal vectors were used for reprogramming peripheral blood mononuclear cells to obtain integration-free iPSC. Cells were collected from patients treated with hydroxyurea and without treatment. The iPSCP Bscd lines were characterized for pluripotent and differentiation potential. The iPSC lines were differentiated into HSC, so that we obtained a dynamic and efficient protocol of CD34+CD45+ cells production. We offer a valuable tool for a better understanding of how SCA occurs, in addition to making possible the development of more effective drugs and treatments and providing better understanding of widely used treatments, such as hydroxyurea