A 10376 bp deletion of FECH gene responsible for erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP, MIM 177000) is an autosomal dominant disease with incomplete penetrance since the phenotypic expression requires coinheritance of a null allele and a wild-type low expressed allele of Ferrochelatase gene (FECH). In this study, we identify a peculiar mutation in a young Canadian patient of Italian origin. The patient had clinical and biochemical symptoms of EPP, the wild-type low expressed allele but at preliminary analysis no mutation in the promoter, in the entire coding region and in the splice junctions of the gene. Family studies of seven most common polymorphisms along the gene established absence of Mendelian segregation for the promoter polymorphism only. The intron 1 polymorphism appeared in heterozygosis suggesting an hypothetical deletion in the first region of the gene. In order to identify the size of this deletion, single nucleotide polymorphisms (SNPs) analysis was extended to the upstream N-asparaginyl-tRNA synthetase gene (NARS). We analyzed two polymorphisms in the last exon of this gene and a dizigous region was found in the patient. A Long-PCR with primers located in previously fixed heterozygous regions showed a 10,376 bp deletion (c.1-7887_67+2422del) that included a portion of the upstream intergenic region, the promoter, the exon 1 and a portion of intron 1. RNA analysis demonstrated that the lack of the entire promoter prevents the expression of the mutated allele, in fact the expression of the Ferrochelatase gene was decreased by half in the subjects carrying only the mutation compared to control.

Expression of the gamma-globin gene is sustained by the cAMP-dependent pathway in beta-thalassaemia.

The present study found that the cyclic adenosine monophosphate (cAMP)-dependent pathway efficiently induced gamma-globin expression in adult erythroblasts, and this pathway plays a role in gamma-globin gene (HBG) expression in beta-thalassaemia. Expression of HBG mRNA increased to about 46% of non-HBA mRNA in adult erythroblasts treated with forskolin, while a cyclic guanosine monophosphate (cGMP) analogue induced HBG mRNA to levels <20% of non-HBA mRNA. In patients with beta-thalassaemia intermedia, cAMP levels were elevated in both red blood cells and nucleated erythroblasts but no consistent elevation was found with cGMP levels. The transcription factor cAMP response element binding protein (CREB) was phosphorylated in nucleated erythroblasts and its phosphorylation levels correlated with HBG mRNA levels of the patients. Other signalling molecules, such as mitogen-activated protein kinases and signal transducers and activators of transcription proteins, were phosphorylated at variable levels and showed no correlations with the HBG mRNA levels. Plasma levels of cytokines, such as erythropoietin, stem cell factor and transforming growth factor-beta were increased in patients, and these cytokines induced both HBG mRNA expression and CREB phosphorylation. These results demonstrate that the cAMP-dependent pathway, the activity of which is augmented by multiple cytokines, plays a role in regulating HBG expression in beta-thalassaemia.

cAMP differentially regulates gamma-globin gene expression in erythroleukemic cells and primary erythroblasts through c-Myb expression.

Our previous studies demonstrated roles of cyclic nucleotides in gamma-globin gene expression. We recently found that, upon activation of the cAMP pathway, expression of the gamma-globin gene is inhibited in K562 cells but induced in adult erythroblasts. Here we show that c-Myb, a proto-oncogene product that plays a role in cell growth and differentiation, is involved in the cAMP-mediated differential regulation of gamma-globin gene expression in K562 cells and primary erythroblasts. Our studies found that c-Myb is expressed at a high level in K562 cells compared to primary erythroblasts, and that c-Myb expression is further increased following the treatment with forskolin, an adenylate cyclase activator. The induction of gamma-globin gene expression was also inhibited in K562 cells by raising the levels of c-Myb expression. Importantly, forskolin-induced erythroid differentiation in K562 cells, as determined by the expression of glycophorins and CD71, suggesting that high-level expression of c-Myb may not be sufficient to inhibit the differentiation of erythroid cells. In contrast, c-Myb was not expressed in adult erythroblasts treated with forskolin and primary erythroblasts may lack the c-Myb-mediated inhibitory mechanism for gamma-globin gene expression. Together, these results show that the cAMP pathway blocks gamma-globin gene expression in K562 cells by increasing c-Myb expression and c-Myb plays a role in defining the mode of response of the gamma-globin gene to fetal hemoglobin inducers in erythroid cells.