ABSTRACT
Individual variation in alcohol consumption in human populations is determined by genetic, environmental, social and cultural factors. In contrast to humans, genetic contributions to complex behavioral phenotypes can be readily dissected in Drosophila, where both the genetic background and environment can be controlled and behaviors quantified through simple high-throughput assays. Here, we measured voluntary consumption of ethanol in â¼3000 individuals of each sex from an advanced intercross population derived from 37 lines of the Drosophila melanogaster Genetic Reference Panel. Extreme quantitative trait loci mapping identified 385 differentially segregating allelic variants located in or near 291 genes at P < 10-8 . The effects of single nucleotide polymorphisms associated with voluntary ethanol consumption are sex-specific, as found for other alcohol-related phenotypes. To assess causality, we used RNA interference knockdown or P{MiET1} mutants and their corresponding controls and functionally validated 86% of candidate genes in at least one sex. We constructed a genetic network comprised of 23 genes along with a separate trio and a pair of connected genes. Gene ontology analyses showed enrichment of developmental genes, including development of the nervous system. Furthermore, a network of human orthologs showed enrichment for signal transduction processes, protein metabolism and developmental processes, including nervous system development. Our results show that the genetic architecture that underlies variation in voluntary ethanol consumption is sexually dimorphic and partially overlaps with genetic factors that control variation in feeding behavior and alcohol sensitivity. This integrative genetic architecture is rooted in evolutionarily conserved features that can be extrapolated to human genetic interaction networks.
Subject(s)
Alcohol Drinking/genetics , Gene Regulatory Networks , Quantitative Trait Loci , Animals , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Genetic Background , Male , Polymorphism, Single Nucleotide , Sex FactorsABSTRACT
Gaucher disease is an excellent candidate for gene therapy by transduction of hematopoitic stem cells. In this study, we compared methods which allow an increase in transfer of the glucocerebrosidase gene to human hematopoietic progenitor cells. Several techniques were employed, including the use of cytokines, bone marrow stroma, fibronectin, centrifugal enhancement and in vitro long-term culture. The effect of prestimulation with cytokines interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) on transduction of cord blood CD34+ cells was examined. The results suggest that 16-h prestimulation was sufficient for efficient transduction. We examined the effect of bone marrow stroma and fibronectin, both of which increased transduction efficiency up to 36% and 44%, respectively, as measured by PCR for the integrated GC-cDNA in clonogenic cells (9% without any support). Transduction efficiency of 83% was obtained using 2-h centrifugation. Combining centrifugation and in vitro culture in long-term bone marrow culture media containing cytokines (IL-3/IL-6/SCF), CD34+ cells from cord blood and peripheral blood of 3 Gaucher patients were transduced weekly for 21 d. The results of 6 separate experiments consistently demonstrated transduction efficiency of 100% after 7-d in vitro culture. This transduction protocol combining centrifugation and in vitro long-term culture is an attractive method and can be applied to clinical trials.
Subject(s)
Antigens, CD34/analysis , Gene Transfer Techniques , Genetic Vectors/blood , Glucosylceramidase/genetics , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/immunology , Retroviridae/genetics , Transfection/methods , Bone Marrow Cells/cytology , Cells, Cultured , Centrifugation , Culture Media , Fibronectins/pharmacology , Gaucher Disease/genetics , Hematopoietic Stem Cells/drug effects , Humans , Interleukin-3/pharmacology , Interleukin-6/pharmacology , Stem Cell Factor/pharmacology , Stromal Cells , Time FactorsABSTRACT
Transfer of the gene coding for glucocerebrosidase (GC) via a retroviral vector (MFG-GC) to haematopoietic progenitors results in engraftment and life-long expression of the human protein at high levels in transplanted mice. Studies of human CD34 cells were carried out to evaluate their potential use in a gene therapy approach to Gaucher's disease. High transduction efficiency and correction of the enzyme deficiency was possible in CD34 cells obtained from patients with Gaucher's disease. Based on these results, a clinical trial of gene therapy was designed and initiated. Preliminary results of this study indicate the persistence or engraftment of genetically corrected cells in the transplanted patients.