Methionine synthase reductase (MTRR) A66G polymorphism is not related to plasma homocysteine concentration and the risk for vascular disease.

Epidemiological evidence has revealed that hyperhomocysteinemia increases the risk for vascular disease. Methionine Synthase Reductase (MTRR) is one of several key enzymes in the homocysteine metabolic pathway and its mutant forms have been implicated in abnormal homocysteine accumulation. In this study, we determined total plasma homocysteine levels and MTRR A66G polymorphism in 114 patients with vascular disease: 58 patients with deep-vein thrombosis, 56 patients with arterial thrombosis, and 95 healthy subjects from the Sicilian population. Our data confirmed that, as already reported, moderately elevated t-Hcy levels are correlated with an increased risk of vascular disease. In our study, the levels of t-Hcy found in both deep-vein thrombosis (13.7+/-3.2 micromol/L) and arterial thrombosis (14.3+/-4.3 micromol/L) patient groups were higher than levels detected in normal subjects (8.7+/-2.7 micromol/L). We concluded that the MTRR A66G polymorphism was not associated with the t-Hcy plasma concentration because the same genotype frequency distribution was detected in both patients and healthy individuals.

A region upstream of the human delta-globin gene shows a stage-specific interaction with globin promoters in erythroid cell lines.

We previously showed that the 651-bp DNA fragment, located 3 kb upstream from the human delta-globin gene (fragment F5), is able to inhibit adult, not fetal, globin promoter in mouse erythroleukemia cell lines (MEL) expressing adult globin genes. Here we show in transient analysis that fragment F5 has a strong inhibitory effect on fetal gamma-globin promoter in human erythroleukemia cell lines (HEL) expressing fetal globin genes. Since the beta-promoter constructs were poorly expressed in fetal cells, new plasmids containing an HPFH promoter (Ggamma(-175), T to C), which is strongly expressed in both fetal and adult cell lines, were made. Here we report that fragment F5 in HEL cells has a strong inhibitory effect on wild-type gamma-promoter only; no effect was evident on gamma(-175)-promoter in either MEL or HEL cell lines. Altogether these results show a stage-specific interaction between fragment F5 and globin promoters during development. We also report the presence of several bindings for erythroid GATA family factors by electrophoretic mobility shift assay, using nuclear extracts from erythroid cell lines.

An element upstream from the human delta-globin-encoding gene specifically enhances beta-globin reporter gene expression in murine erythroleukemia cells.

We have previously shown that a DNA-binding factor specific to adult hematopoietic cells (polypryrimidine-binding factor, PYBF) binds to a pyrimidine-rich region 1 kb upstream from the human delta-globin-encoding gene (HBD). The developmental stage-specificity of PYBF and the location of its binding site between the fetal and adult beta-globin (HBB)-like genes suggest that PBYF and its binding site may function in fetal-to-adult globin gene switching. Here, we describe the effect of 383-bp (delta383) and 99-bp (delta99) sequences containing the PYBF-binding site on transcription from various globin and non-globin promoters, using a transient assay with the cat reporter gene in murine erythroleukemia (MEL) cells, a cell line with abundant PYBF activity. We show that both delta383 and delta99 specifically enhance expression of cat for plasmids containing a human adult globin (HBB) promoter, whereas expression of similar constructs using human fetal (A gamma-) globin (HBG1) or simian virus 40 (SV40) promoters is not enhanced. The results suggest that PYBF and the pyrimidine-rich region upstream from HBD can specifically enhance HBB transcription in adult erythroid cells.

Evidence for a globin promoter-specific silencer element located upstream of the human delta-globin gene.

We describe the negative regulatory activity of a 1.7 kilobase (kb) region (R) in the human beta-globin locus located between 4.0 and 2.3 kb upstream of the delta-globin gene capsite, using a transient assay with the chloramphenicol acetyltransferase (CAT) reporter gene in mouse erythroleukemia (MEL) cells. The R region is deleted in most cases of deletion hereditary persistence of fetal hemoglobin (HPFH), but is unaffected in most delta beta zero-thalassemias. However, no experiments addressing its function in globin gene expression have been reported to date. We show that R inhibits CAT expression of constructs containing a fetal (gamma) or adult (beta) globin gene promoter, but does not affect expression of similar constructs using a non-globin (SV40) promoter. The inhibitory effect on the beta-globin promoter can be localized to a 651 bp sub-region of R. For the gamma-globin promoter, no sub-region of R can reproduce the level of inhibition associated with the entire region.

Amplification of ETS2 oncogene in acute nonlymphoblastic leukemia with t(6;21;18).

Cytogenetic and molecular studies in a case of acute nonlymphoblastic leukemia (ANLL) are reported in this paper. Bone marrow blasts carried a hypodiploid karyotype with a complex t(6;18;21)(6qter----6p21::21q22----21qter;18qter ----18p11::6p22----6pter; 21pter----21q22::6p21----6p22::18p11----18pte r) and other numerical and structural changes. We studied the organization and the expression of the ETS2 gene which is located on chromosome 21 in order to investigate its possible involvement in the disease. DNA analysis showed a 20-fold amplification of ETS2 sequences; an increase of 3- to 4-fold in the mRNAs level compared to normal was shown by Northern hybridization.

The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562.

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3' enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3' enhancer is erythroid-specific but not developmental stage- or gene-specific.

The regulation of gamma-globin gene expression.

In summary, our analysis indicates that important sequences for the proper initiation of fetal gene transcription in fetal cells are located in the gamma-globin [sequence: see text] promoter. These sequences are sufficient for tissue-specific expression but not induction in K562 cells. Sequences in the gamma-globin IVS-2 and the beta-globin 3' enhancer increase gamma beta and gamma-Neo transcripts when cells containing these genes undergo erythroid maturation as measured by induction with hemin. The mechanism by which these sequences exert their effect remains to be elucidated. [see text] Multiple protein factors bind to both the gamma promoter and the beta 3' enhancer. Both of these regions contain binding sites for the erythroid-specific factor NFE-1 and the octamer binding factor OTF-1. In the gamma upstream region, there may be a competition between OTF-1 binding and NFE-1 binding that affects gamma gene regulation. Our results indicate that the beta 3' enhancer interacts with the gamma gene promoter to permit increased gamma gene expression. We have developed a model for globin gene switching that takes into consideration the effect of cis-acting sequences on globin gene transcription. A similar model of hemoglobin switching in chickens has been proposed by Choi and Engel. In our model, competition for the beta-globin 3' enhancer is involved in stage-specific transcriptional activation of gamma-globin genes in fetal cells and beta-globin genes in adult cells. In adult cells the protein-protein interactions between adult cell-specific factors interacting with the beta-globin promoter and erythroid-specific factors interacting with the beta 3' enhancer would activate transcription of the beta-globin gene. In fetal cells protein-protein interactions between fetal cell-specific factors interacting with the gamma-globin promoter and erythroid-specific factors interacting with the beta 3' enhancer would activate the transcription of the gamma-globin genes.

Regulation of human fetal hemoglobin gene expression.

An understanding of the mechanism involved in the regulated expression of the human gamma and beta globin genes requires the detailed definition of the cis-acting DNA sequences and trans-acting protein factors responsible for their developmental stage specific expression. To determine the critical cis-acting elements, hybrid genes containing elements of the gamma and beta globin genes were transfected into K562 cells, a human erythroleukemia line. The regulated expression of the gamma and beta genes was also studied by transferring hybrid genes containing the gamma or beta promoters linked to the neomycin resistance gene (neoR) into erythroid (K562) cells and nonerythroid (Hela) cells. DNA sequences found to be important to the expression of the gamma gene were assayed for the presence of transacting factors by studying the binding of protein factors using the gel mobility shift assay. The results suggest that there are multiple cis-acting elements 5' and 3' to the gamma and beta genes, and perhaps within these genes contributing to their regulation. In addition, there are multiple trans-acting protein factors interacting with these regions which may determine their transcriptional regulation in erythroid cells.

Regulation of fetal globin gene expression in human erythroleukemia (K562) cells.

We have analyzed the transcription and induction of fusion globin genes comprised of portions of either gamma and beta globin sequences or gamma and neomycin resistance gene sequences. The analysis of gamma promoter beta and gamma-neo fusion genes indicates that 5' gamma flanking sequences are sufficient for tissue specific expression but not induction in K562 cells. A beta gene containing only the substitution of gamma IVS 2 for beta IVS 2 is expressed and induced when transcripts are analyzed with a 3' probe in contrast to the lack of expression seen with an intact beta gene. Thus, fusion globin genes containing gamma IVS 2 are both expressed and induced indicating that this region may be involved in the response to hemin stimulation, however, the mechanism is unclear. A gamma-neo fusion gene containing the gamma 5' region is expressed but not induced. When an EcoRI-Bg1 II fragment containing the beta 3' enhancer is ligated downstream of the gamma-neo gene this gene is now inducible. Multiple genetic elements are involved in the regulated expression of gamma genes in fetal erythroid cells. These experiments begin to localize these sequences to specific regions within the gamma globin gene.

Expression of human gamma-globin genes in human erythroleukemia (K562) cells.

K562 cells express embryonic (epsilon) and fetal (gamma) globins and hemoglobins but not adult (beta) globin. To define the cis acting regulatory elements involved in the discrimination between gamma and beta genes, we have constructed chimeric genes composed of portions of gamma and beta and evaluated their expression in stable K562 transfectants. A gamma beta fusion gene containing gamma 5' sequences to the EcoRI site in exon 3 and beta sequences 3' is expressed at 10-40% that of the endogenous gamma level. In 50% of the lines, this fusion gene appropriately increases its expression in response to hemin, an inducer of endogenous globin gene expression in K562 cells. In contrast, a beta gamma fusion gene, containing beta sequences 5' to the EcoRI site in exon 3 and gamma sequences 3', is neither expressed nor correctly initiated. A beta gene containing gamma-intervening sequence (IVS) 2 accumulates an mRNA transcript when analyzed with a 3' beta probe. However, no correctly initiated beta mRNA is observed. A gamma gene with beta-IVS 2 is only inducible in one of six expressing clones. All the results are consistent with the presence of stage-specific trans acting factors in K562 cells that stimulate expression of gamma genes and suggest a significant role for gamma-IVS 2 in gamma gene expression.

Expression of a beta thalassemia gene with abnormal splicing.

Expression of a cloned human beta thalassemia gene with a single base change at position 5 of IVS 1 has been analyzed 48 hours after transfer of the gene into HeLa cells (transient expression). Little or no normal beta globin mRNA accumulates in the presence of the abnormal beta gene in contrast to significantly more normal beta mRNA produced with other mutations at this same position. By contrast, large amounts of an abnormal beta globin mRNA are present; this is due to the use of a cryptic 5' splice site in exon 1 rather than the normal 5' splice site of IVS 1. The results indicate the variability of the effect on RNA splicing of different single base defects within IVS.

Expression of chimeric human beta- and delta-globin genes during erythroid differentiation.

To determine whether sequences contained within the small intervening sequence (IVS 1) or large intervening sequence (IVS 2) are involved in the regulated expression of the human beta-globin gene, chimeric genes containing portions of the human beta- and delta-globin genes were stably transfected into mouse erythroleukemia (MEL) cells. Since MEL cells can be induced to differentiate in culture, the expression of the chimeric genes was compared to the expression of beta and delta both before and after the induction of erythroid differentiation. The expression of beta delta 1, a beta-globin gene containing delta IVS 1 in place of beta IVS 1, was comparable to the expression of a beta-globin gene both before and after erythroid differentiation. However, the base-line expression of human beta-globin genes containing delta IVS 2 in place of beta IVS 2 was dramatically decreased. Furthermore, the substitution of delta IVS 2 for beta IVS 2 prevented the regulated increase in expression of the beta-globin gene upon induction. The results also indicate that sequences present in beta IVS 2 are not sufficient for this induced increase in expression since the substitution of beta IVS 2 for delta IVS 2 in a delta gene does not increase the regulated expression of delta during differentiation. These experiments suggest that either the presence of delta IVS 2 in a beta gene interrupts sequences required for the induced expression of beta-globin or that sequences in beta IVS 2 act in concert with other beta globin sequences not present in the delta-globin gene to permit optimal expression.

The role of human globin gene promoters in the expression of hybrid genes in erythroid and non-erythroid cells.

Hybrid genes containing human gamma or beta globin gene promoters linked to a neomycin resistance (neoR) gene were transfected into erythroid (K562) and nonerythroid (HeLa) cells. The number of clones resistant to G418, a neomycin analogue, was used to assay promoter strength. The results indicate that in K562 cells both promoters are active, and the gamma gene promoter is much stronger than the beta. By contrast, neither gene promoter is active in HeLa cells. These experiments indicate that these globin gene promoters are tissue-specific and sufficient for activity.

Beta A and beta thal DNA haplotypes in Sicily.

A close association between specific restriction fragment polymorphism patterns and specific mutations in Mediterranean people with thalassemia has been demonstrated by Kazazian et al. (1984). This finding is useful to characterize the number and types of mutations in each ethnic group for setting up prenatal diagnosis in the first trimester of pregnancy by the oligonucleotide technique. For this reason we studied 99 beta thal and 46 beta A chromosomes in the Sicilian population. We found seven different cleavage patterns, not considering two new haplotypes so far uncharacterized. Many of the patients (68.3%) were genetic compounds for different haplotypes while only 31.7% were haplotype homozygotes. They may still be thalassemia compound heterozygotes. These findings confirm the molecular basis of the heterogeneity of beta thalassemia in Sicily.

Clinical severity of non-deletion form of HbH disease (--Med/alpha alpha thal).

We carried out alpha-globin gene analysis by restriction endonuclease mapping in a family with 2 cases of HbH disease. These data show that HbH disease in this family results from the interaction between a common deletional defect and a less common non-deletion alpha-thal lesion (--Med/alpha alpha thal). Furthermore, the presence of a beta-thal determinant in this family was investigated by beta gene polymorphism study. We showed that a patient with HbH disease also inherited a beta-thal determinant from the mother and although this was a beta O-thal gene, it was not sufficient to mask the severe alpha chain deficiency. The --Med/alpha alpha thal genotype is more severe than other types of alpha thalassaemia interactions causing HbH disease, probably because the expression of alpha alpha thal determinant may be lower than that of an alpha-thal determinant containing just a single alpha gene (-alpha) and the output so poor that the presence of one beta-thal gene does not significantly change the clinical picture.