ABSTRACT
A considerable progress has been made in the last three years in the uncovering of the molecular basis of Diamond-Blackfan anaemia (DBA). Two genetic loci on 19q13.2 and 8p23 chromosomes have been associated with the DBA phenotype, and the ribosomal protein S19 (RP S19) located at 19q has been found mutated in 25% of DBA patients. In this review we will outline possible mechanisms of how mutations in RP S19 might lead to the DBA phenotype, we will discuss candidate genes on 8p23 chromosome, and finally, a complex molecular model of DBA development will be proposed.
Subject(s)
Anemia, Diamond-Blackfan/genetics , Chromosomes, Human, Pair 8/genetics , Erythropoiesis/physiology , Humans , Mutation , Protein Biosynthesis , Ribosomal Proteins/genetics , Ribosomal Proteins/physiologyABSTRACT
The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy. However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA. We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA. Sequence alterations of the cSD reduced the expression of MDR1 P-glycoprotein (P-gp), even when generated as silent mutations. A silent mutation of the cSA reduced the splicing activity shifting the splice acceptor site one base downstream; however, it significantly improved the expression of P-gp. The incidence of wild-type MDR1 pregenome splicing was markedly reduced when vectors were produced in human 293 packaging cells as opposed to murine PG13 and GP+envAm12. We conclude that complete splice correction of MDR1 in retroviral vectors may only be achieved with extensive alterations of the cDNA or neighboring vector sequences and that the splicing is significantly influenced by the choice of the packaging cells.
