Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors.

Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications

Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish f10 by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use f102/2zebrafish to confirm 5 novel human F10 variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of F10 variants. These findings as well as the prolonged survival of f102/2mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders

Loss to follow up from isoniazid preventive therapy among adults attending HIV voluntary counseling and testing sites in Uganda.

Among HIV-infected adults attending non-governmental organization voluntary counseling and testing (VCT) sites in Uganda that provide a nine-month course of isoniazid preventive treatment (IPT), we report on loss to follow-up (LTFU) and its associated risk factors. The design was a retrospective cohort study of program data spanning a three year period (2006-2008). A total of 586 IPT patients were enrolled of whom 335 (57.1%) were females with a mean age of 34 years. Of those starting IPT, 341 (58.1%) were lost to follow-up, 197 (33.6%) completed IPT, 29 (4.9%) were discontinued and 19 (3.2%) died. The return rates at one, three, five and seven months were 78.0% (457), 62.1% (364), 52.9% (310) and 33.6% (197) respectively. Being less than 30 years of age, widowed, separated, or divorced were found to be associated with a higher risk of loss to follow-up. Sudden improvement in retention on IPT was observed between the years 2006 and 2007, although causes of the improvement are poorly understood hence the need for more research. At non-governmental VCT sites in Uganda, six out of ten individuals enrolled on IPT are lost to follow-up and efforts to reduce this attrition including systems strengthening might play a critical role in the success of IPT programs