An initiation codon mutation (AUG----GUG) of the human alpha 1-globin gene. Structural characterization and evidence for a mild thalassemic phenotype.

alpha-globin is encoded by two adjacent genes, alpha 1 and alpha 2. Recent evidence suggests that these genes are not equally expressed and that the alpha 2-globin gene encodes the majority of alpha-globin. This finding would predict that a thalassemic mutation of the alpha 2-globin gene would result in a more severe loss of alpha-chain synthesis than a similar mutation in the alpha 1-globin gene. In a previous study we described a nondeletion alpha-thalassemia defect in the alpha 2-globin gene resulting from an AUG----ACG initiation codon mutation. In the present study we describe a different initiation codon mutation, AUG----GUG, present in the alpha 1-globin gene. The alpha 1- and alpha 2-globin gene initiation codon mutations result in similarly lowered levels of encoded mRNA. Despite the similarity of these two mutations, the alpha 2 mutant results in a more severe loss of alpha-globin synthesis and a more severe clinical alpha-thalassemia phenotype than the corresponding alpha 1-globin gene mutation. This difference reflects the dominant role of alpha 2-globin gene in overall alpha-globin synthesis.

Nuclear factor-erythroid 2 (NF-E2) expression in normal and malignant megakaryocytopoiesis.

Although the transcription factor nuclear factor-erythroid 2 (NF-E2) is known to be functionally linked to the megakaryocytic lineage, little is known about its role in malignant megakaryocytes. We used real-time RT-PCR and Western blotting to investigate expression of NF-E2 and its partner, MafG, in CD34-derived normal (five cases) and malignant megakaryocytes from essential thrombocythemia (ET) patients (eight cases) and in megakaryoblastic cell lines. We also quantitated the mRNA of the thromboxane synthase (TXS) gene, which is directly regulated by NF-E2. Although real-time RT-PCR showed that both a and f NF-E2 isoforms were significantly reduced with respect to the normal counterpart both in ET megakaryocytes and in cell lines (P < or = 0.01), western blotting revealed decreased NF-E2 protein expression only in the latter. However, both the NF-E2a/MafG mRNA ratio (P < or = 0.01) and TXS (P< or = 0.01) mRNA expression were significantly reduced in megakaryocytes from ET patients and cell lines with respect to healthy subjects. These two findings provide strong indirect evidence of altered activity of the a isoform of NF-E2 in malignant megakaryocytes, raising the possibility that NF-E2 could play a role in megakaryocyte transformation.

A novel missense mutation (C84R) in a patient with type II vitamin d-dependent rickets.

A 7-year-old boy with severe rickets that by clinical analysis was diagnosed as affected by type II vitamin D-dependent rickets, was evaluated for mutations in the vitamin D receptor gene (VDR). The molecular analysis showed a homozygous state for a novel missense mutation (C84R) in a highly conserved nucleotide in the second Zn finger of the DNA binding domain.

Genetic modifying factors in beta-thalassemia.

The beta-thalassemia is probably the most extensively studied genetic disease. Essentially any molecular defect that has been first described in association with the globin genes has been later implicated as a molecular determinant of newly discovered genes. Accordingly, the thalassemias have always represented a model genetic disease, especially in relation to the development of programs for population screening, genetic counseling and prenatal diagnosis. Here we will review the present knowledge on the genetics of thalassemia and of the relevant modifying factors. Major categories of the carrier state, the genotypes, the clinical phenotypes and the correlation between genotype and phenotype will be discussed.

Synergistic enhancement of globin gene expression by activator protein-1-like proteins.

DNA sequences corresponding to the four major DNase I hypersensitive sites upstream of the beta-globin gene cluster are essential for the achievement of high levels of globin gene expression and development regulation. In this study, we focused on one of these sites, hypersensitive site 2, which behaves as a powerful enhancer in transient expression and transgenic mouse experiments. We identified a tandem repeat of the activator protein 1 (AP-1) consensus sequence that binds AP-1-like proteins from nuclear extracts of K562 and HeLa cells. These proteins have the same binding properties as HeLa AP-1 but differ in the electrophoretic mobility and in functional assays. Transient-expression experiments in K562 of various deletion and point mutation constructs derived from hypersensitive site 2 indicate that the enhancer activity and the inducibility of a linked gamma-globin promoter are dependent upon the synergistic action of proteins bound to the tandem AP-1 repeat.

hMAF, a small human transcription factor that heterodimerizes specifically with Nrf1 and Nrf2.

A 1.6-kilobase pair full-length cDNA encoding a transcription factor homologous to the Maf family of proteins was isolated by screening a K562 cDNA library with the NFE2 tandem repeat probe derived from the globin locus control region. The protein, which was designated hMAF, contains a basic DNA binding domain and an extended leucine zipper but lacks any recognizable activation domain. Expressed in vitro, the hMAF protein is able to homodimerize in solution and band-shift the NFE2 tandem repeat probe. In addition to homodimers, hMAF can also form high affinity heterodimers with two members of the NFE2/CNC-bZip family (Nrf1 and Nrf2) but not with a third family member, p45-NFE2. Although hMAF/hMAF homodimers and hMAF/Nrf1 and hMAF/Nrf2 heterodimers bind to the same NFE2 site, they exert functionally opposite effects on the activity of a linked gamma-globin gene. In fact, whereas all hMAF/CNC-bZip heterodimers stimulate the activity of a gamma-promoter reporter construct in K562 cells, the association into homodimers that is induced by overexpressing hMAF inhibits the activity of the same construct. Thus variations in the expression of hMAF may account for the modulation in the activity of the genes that bear NFE2 recognition sites.

A homozygous nonsense mutation of the human growth hormone receptor gene in a Sardinian boy with Laron-type dwarfism.

Laron-type dwarfism (LTD) is an autosomal recessive disorder due to mutations in the GH receptor (GHR) gene. We report the case of a Sardinian boy affected by LTD in which we found by direct genomic sequencing a nonsense mutation in the fourth exon of the GHR gene (R43X) that determines a premature termination in the protein translation process. As the result of the absence of the extracellular portion of the GHR this patient had undetectable GH binding protein. This molecular defect is identical to that observed in other patients with LTD of mediterranean origin.

Normal delta-globin gene sequences in Sardinian nondeletional delta beta-thalassemia.

In order to clarify the reasons for the reduced Hb A2 levels in Sardinian delta beta-thalassemia, we characterized, both by cloning and sequence analysis and by direct sequencing of amplified DNA, the delta-globin gene from an individual of Sardinian descent who is a compound heterozygote for the beta zero-thalassemia codon 39 (C-->T) nonsense mutation and the Sardinian delta beta-thalassemia [codon 39(C-->T)/-196(C-->T)A gamma]. The analysis of the delta-globin gene from the delta beta-thalassemia chromosome revealed an entirely normal sequence. The defective function of the delta-globin gene in this determinant is thus likely related to a suppressive effect of the in cis nondeletional high persistence of fetal hemoglobin mutation of the A gamma gene, probably resulting from an increased capability of the relative promoter to interact with the locus control region.

Molecular characterization of a normal Hb A2 beta-thalassaemia determinant in a Sardinian family.

In this study we have carried out haplotype analysis at the beta-globin gene cluster and defined the beta-thalassaemia mutations in a large Sardinian family, ascertained through a proband with thalassaemia major, in which several members were carriers of a beta-thalassaemia allele characterized by microcytosis, hypochromia and normal Hb A2 levels (type 2 normal Hb A2 heterozygous beta-thalassemia). The proband was a compound heterozygote for the beta zero 39 and the beta + IVS-2, nt 745 mutations and all the beta-thalassaemia heterozygotes with normal Hb A2 showed the beta + IVS-2, nt 745 mutation, always associated with haplotype VII. Because of the consistent association of a specific beta-thalassaemia mutation and normal Hb A2 levels, we postulate that this beta-thalassaemia chromosome carries a delta gene (delta-thalassaemia) which is unable to increase the delta-globin output in response to beta-thalassaemia.

Molecular pathology of haemoglobin H disease in Sardinians.

We investigated the molecular basis for haemoglobin H disease in 50 Sardinian patients by restriction endonuclease analysis. We found that the majority (78% of the cases) are due to gene deletion (- -/- alpha). Among those with a combination of deletion and nondeletion defects (- -/alpha alpha th), the most prevalent nondeletion lesion (70% of the nondeletion defects) was the initiation codon mutation of the alpha 2 gene (alpha Nco alpha), previously discovered in this population. Of the remaining patients with the (- -/alpha alpha th) genotype, two showed the IVS-1 splice junction lesion and one a mutation in the alpha 1 gene, removing the Nco I site within the 5' part of the alpha 1 gene, which may arise from a process of gene conversion from the initiation codon mutant of the alpha 2 gene. A single patient had the homozygous state for the initiation codon mutant of the alpha 2 gene. Study of genotype-phenotype correlations indicates that the (alpha Nco alpha) haplotype is associated with a more severe defect in the alpha-globin chain output than that resulting from the (-alpha) haplotype. We may conclude that restriction endonuclease analysis is a powerful method for the definition of the molecular heterogeneity of haemoglobin H disease.

Phenotype-genotype correlation in haemoglobin H disease in childhood.

In this study we used restriction endonuclease mapping to characterise the molecular defect responsible for haemoglobin H disease in 14 Sardinian children. The resulting genotypes were then correlated with the respective clinical and haematological phenotypes. We found that patients with the combination of non-deletion alpha(+)-thalassaemia [(alpha alpha)th] and deletion alpha(0)-thalassaemia (-Med) have a more severe phenotype than that resulting from the interaction of deletion alpha(0)-thalassaemia (-Med) and alpha(+)-thalassaemia (-alpha) determinants. Clinically, presentation was earlier and with moderate anaemia or haemolytic crisis, enlargement of the liver and spleen, and thalassaemic bone changes. Haematologically, the anaemia was more severe and there were higher bilirubin levels, reticulocyte counts, Hb H levels, and percentage of red blood cells with inclusion bodies. These results suggest that in those Hb H disease patients with the non-deletion [(alpha alpha)th] determinant, two alpha globin genes produce fewer alpha globin chains than a single alpha globin locus.

Regulation of mouse p45 NF-E2 transcription by an erythroid-specific GATA-dependent intronic alternative promoter.

The erythroid-enriched transcription factor NF-E2 is composed of two subunits, p45 and p18, the former of which is mainly expressed in the hematopoietic system. We have isolated and characterized the mouse p45 NF-E2 gene; we show here that, similar to the human gene, the mouse gene has two alternative promoters, which are differentially active during development and in different hematopoietic cells. Transcripts from the distal promoter are present in both erythroid and myeloid cells; however, transcripts from an alternative proximal 1b promoter, lying in the first intron, are abundant in erythroid cells, but barely detectable in myeloid cells. During development, both transcripts are detectable in yolk sac, fetal liver, and bone marrow. Transfection experiments show that proximal promoter 1b has a strong activity in erythroid cells, which is completely dependent on the integrity of a palindromic GATA-1 binding site. In contrast, the distal promoter 1a is not active in this assay. When the promoter 1b is placed 3' to the promoter 1a and reporter gene, in an arrangement that resembles the natural one, it acts as an enhancer to stimulate the activity of the upstream promoter la.

Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region.

Hypersensitive site 2 located in the beta-globin locus control region confers high levels of expression to the genes of the beta-globin cluster. A tandem repeat of the consensus sequence for the transcription factors AP1 and NF-E2 (activating protein 1 and nuclear factor erythroid 2, respectively) is present within hypersensitive site 2 and is absolutely required for strong enhancer activity. This sequence binds, in vitro and in vivo, to ubiquitous proteins of the AP1 family and to the recently cloned erythroid-specific transcription factor NF-E2. Using the tandem repeat as a recognition site probe to screen a lambda gt11 cDNA expression library from K562 cells, we isolated several DNA binding proteins. Here, we report the characterization of one of the clones isolated. The gene, which we named Nrf2 (NF-E2-related factor 2), is encoded within a 2.2-kb transcript and predicts a 66-kDa protein with a basic leucine zipper DNA binding domain highly homologous to that of NF-E2. Although Nrf2 is expressed ubiquitously, a role of this protein in mediating enhancer activity of hypersensitive site 2 in erythroid cells cannot be excluded. In this respect, Nrf2 contains a powerful acidic activation domain that may participate in the transcriptional stimulation of beta-globin genes.

Chromosomal localization of the human NF-E2 family of bZIP transcription factors by fluorescence in situ hybridization.

A family of human genes encoding basic-leucine zipper (bZIP) transcription factors, p45-NF-E2, Nrf1 and Nrf2, have been isolated independently. Whereas the encoded proteins of the three genes share highly conserved regions distinct from other bZIP families such as Jun or Fos, remaining regions diverged considerably from each other. Chromosomal localization by fluorescence in situ hybridization demonstrates that these genes are non-syntenic. p45-NF-E2 mapped to chromosome 12q13.1-13.3, whereas Nrf1 and 2 mapped to 17q21.3 and 2q31, respectively. However, these three genes were probably derived from a single ancestor by chromosomal duplication as other genes that also map in these regions are related to one another.

Interaction of heterozygous beta zero-thalassemia with single functional alpha-globin gene.

In this study, we analyzed the phenotypic manifestations resulting from the interaction of heterozygous beta zero-thalassemia(beta zero-39 nonsense mutation) with the functional loss of three alpha-globin structural genes in six subjects, of whom four had the [-alpha/--]alpha-globin genotype and two the [--/alpha Th alpha] alpha-globin genotype. The beta-thalassemia defect was in all cases the nonsense mutation at codon 39. The nondeletion alpha-thalassemia alpha th was the initiation codon mutation (AUG----GUG) of the alpha-2 gene. In all these subjects hypochromia and microcytosis were more marked than in beta zero-thalassemia heterozygotes with a full complement of four alpha-globin genes. All but one had moderate anemia. The alpha:beta globin chain synthesis ratios were consistently decreased. No cases had Hb H on electrophoresis. Subjects with [--/alpha Th alpha] alpha-globin genotype had more severe thalassemia-like manifestations than those with [--/-alpha] alpha-globin genotype.

Delineation of the molecular basis of delta- and normal HbA2 beta-thalassemia.

In this study, we used cloning and sequence analysis to define the molecular defect in two delta-thalassemia genes, one associated with reduced output of delta-globin chains (delta +thal) from a Sardinian and the other with a complete suppression of delta-chain production from the affected locus (delta zerp thal) from a Southern Italian. Sequence analysis of the delta +thal gene showed a G----T substitution at the first nucleotide of codon 27 (delta +27) which produces an amino acid change (Ala----Ser) and presumably activates a cryptic splice site located at this position. Therefore, only a fraction of the transcript is processed from this site, as indicated by the clinical phenotype of delta +thal. DNA sequencing of the delta zero thal gene revealed a T----C substitution at position 1 of IVS-1, which abolishes the splicing at this site and thus leads to complete deficiency of normal mRNA explaining the clinical phenotype of delta zero thal. Oligonucleotide analysis was used to confirm the coinheritance of the delta +27 mutation in a group of Sardinians with thalassemia like phenotype and normal HbA2 level who, on the basis of genetic criteria, were supposed to be double heterozygous for delta-thalassemia and beta-thalassemia. The definition of delta-thalassemia defects in each high-risk area facilitates identification of double heterozygotes for delta- and beta-thalassemia by DNA analysis and may thus improve genetic counseling.

Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice.

The beta-globin locus control region (LCR) consists of four erythroid-specific DNase I-hypersensitive sites, which are necessary for high-level expression of the beta-like globin genes in erythroid tissues. One of these sites, designated 5'HS-2, functions as an erythroid-specific enhancer element in transfection and transgenic mouse experiments. Recent transfection experiments and studies of DNA-protein interactions have localized the 5'HS-2 enhancer to 18 nucleotides that contain a binding site for both the erythroid-specific factor nuclear factor erythroid 2 (NFE-2) and for activator protein 1 (AP-1). To define the sequences necessary for in vivo enhancer activity, several deletion mutants of 5'HS-2 were linked to the human beta-globin gene and their activity was tested in transgenic mice. Three upstream fragments of 5'HS-2 [341, 374, and 412 base pairs (bp)], each of which contained the NFE-2/AP-1 sequences, resulted in beta-globin expression at levels equivalent to or higher than those observed with the entire 732-bp 5'HS-2 fragment. In contrast, a 358-bp downstream portion of 5'HS-2, which lacked the NFE-2/AP-1 sequences, resulted in beta-globin expression at the low levels seen with the beta-globin gene alone. Removal of the NFE-2/AP-1 sequences by a 67-bp internal deletion resulted in similar low levels of beta-globin expression. A 100-bp 5' fragment that contained the NFE-2/AP-1 sequences resulted in beta-globin expression that was higher than the beta-globin gene alone but lower than the entire 5'HS-2 fragment or the three larger upstream fragments. These studies demonstrate that the NFE-2/AP-1 sequences are essential for enhancer activity of 5'HS-2 but that other sequences are required for full activity in vivo.

Inter-ethnic polymorphism of the beta-globin gene locus control region (LCR) in sickle-cell anemia patients.

Sequence polymorphisms within the 5'HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major beta s haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.

Isolation of a differentially regulated splicing isoform of human NF-E2.

The transcription factor NF-E2 (nuclear factor erythroid 2), interacting via DNA motifs within regulatory regions of several hematopoietic genes, is thought to mediate the enhancer activity of the globin locus control regions. By screening a human fetal liver cDNA library with probes derived from mouse NF-E2, we have isolated a splicing variant of the NF-E2 gene (fNF-E2) that differs in the 5' untranslated region from the previously reported cDNA (aNF-E2). The fNF-E2 isoform is transcribed from an alternative promoter located in the 3' end of the first intron and joined by alternative splicing to the second and third exons, which are shared by both RNA isoforms. Although the two forms produce the same protein, they are expressed in different ratios during development. fNF-E2 is more abundant in the fetal liver and less abundant in the adult bone marrow compared to the previously described form. Their distribution apparently follows the differential expression of fetal and adult hemoglobins.

A novel splicing defect (IVS6+1G>T) in a patient with pseudovitamin D deficiency rickets.

A 15-month-old boy with severe rickets, that by clinical analysis was diagnosed as affected by hereditary pseudovitamin D deficiency rickets (PDDR), was evaluated for mutations in the 25OHD3 1alpha-hydroxylase gene. Molecular analysis showed a double heterozygous state for a novel splicing mutation in the invariant dinucleotide of the donor site of IVS6 and a 7 nucleotide insertion in the exon 8, which is common in different ethnical backgrounds.