Barbara Giglioni

Globin gene deletion in HPFH, δ°β° thalassaemia and Hb Lepore disease

THE thalassaemias are a group of inherited disorders characterised by the defective production of either α (α thalassaemias) or non-α (β and δ°β° thalassaemias) globin chains of haemoglobins (Hb)1. In β thalassaemias the decreased synthesis of β-globin chains is only partially compensated by the increased production of γ chains, which probably reflects2 the massive hypertrophy of the erythron with selective survival of the clones of adult haemoglobin F-producing cells (F cells3,4). The situation is very different in other genetic disorders of the non-α gene cluster, known as δ°β° thalassaemias and Negro type of hereditary persistence of fetal haemoglobin (HPFH). In these two forms there is a genuine increase of γ-chain production, as shown by the high level of HbF found in heterozygotes. Although a clearcut distinction from both the clinical and haematological point of view cannot be traced between these two forms, the HPFH differs from the δ°β° thalassaemia in having a higher degree of γ-chain synthesis and a more homogeneous distribution of HbF within red cells. Recently, it has been possible to carry out gene analysis on DNA prepared from β°, δ°β° thalassaemic and HPFH patients. The β-globin gene is present in β° thalassaemias5–9, but in δ°β° thalassaemias and HPFH a major deletion, possibly involving both δ and β genes, has been demonstrated by hybridisation studies8,10–12. To characterise the molecular defect in these genetic disorders more precisely, we have hybridised DNA from homozygotes with HPFH, δ°β° thalassaemia and Hb Lepore disease (in which non-α-chains are a δβ fusion product13). For this we used a pure full-size cDNAβ probe and specific 5′ end and 3′ end cDNA fragments (we designate as 5′ end cDNA the portion corresponding to the 5′ end of the mRNA; the same for the 3′ end). Our results, reported here, show that in contrast to HPFH, where a complete δ and β gene deletion occurs, in δ°β° thalassaemia a 5′-end fragment of the δ gene is present.

Gγ and Aγ globin chains separation and quantitation by isoelectric focusing

Abstract Isoelectric focusing in the presence of Nonidet P-40 splits human chromatographically pure γ globin chains into two bands of isoelectric points 6.95 and 6.85, respectively. The comparison of the relative proportions of the two bands with the ratios between the Gγ and Aγ non allelic chains of human fetal hemoglobin suggests that the band at pI 6.95 corresponds to Gγ and the band at pI 6.85 corresponds to the Aγ chain; the latter is the only band present in a patient with Greek type hereditary persistence of fetal hemoglobin, producing only Aγ chains. Fluorography of electrofocusing-separated radioactive γ globin chains synthesized by thalassemic reticulocytes indicates that the relative G γ A γ synthetic ratios are similar to the relative amounts of Gγ and Aγ chains accumulated in the erythrocytes, suggesting that the activities for the Gγ and Aγ mRNAs decay at roughly similar rates.

A direct estimate of the number of human γ-globin genes

Abstract The number of genes specifying human γ-globin has been determined directly by hybridization of complementary DNA to total human DNA. The complementary DNA was enriched in sequences specific for γ-globin genes by transcribing globin mRNA isolated from fetal reticulocytes with viral reverse transcriptase, and collecting the material which does not back-hybridize to adult globin mRNA. When hybridized in cDNA excess to DNA, very similar values are found for γ-gene number as for β-gene number, suggesting two or at most three γ-globin genes per haploid human genome. This indicates that the non-Mendelian ratios of γ-chain mutants found in heterozygotes are due to transcriptional or post-transcriptional regulation rather than to gene dosage. The number of each major human globin gene has now been determined directly by molelcular methods.

DNA Sequences Regulating Human Globin Gene Transcription in Nondeletional Hereditary Persistence of Fetal Hemoglobin

Strong genetic evidence supports the idea that point mutations in the promoter of gamma-globin genes overexpressed in adult age [hereditary persistence of fetal hemoglobin (HPFH)] are responsible for the observed phenotype. DNA binding sites for ubiquitous and/or erythroid specific nuclear proteins correlate in location with the positions of point mutations responsible for HPFH. The analysis of the effects of one of these mutations (-175 T greater than C) on in vitro binding of nuclear proteins and on the activity of the mutated promoter in transfection assays indicates that altered binding of the erythroid-specific protein NFE-1 may be responsible for increased activity of the mutated promoter. Other HPFH mutations close to the distal CCAAT box (-117 G greater than A and 13 nucleotide deletions, -114 to -102) have complex effects on in vitro binding of nuclear proteins; their only common effect is the loss of binding of the erythroid-specific factor NFE3. If mechanisms generating the HPFH phenotype are homogeneous, NFE3 might be a negatively acting factor; alternatively, heterogeneous mechanisms might operate and HPFH might additionally be related to loss of binding to the distal CCAAT box region of either NFE1 (-117 HPFH) or of the ubiquitous CCAAT displacement protein-CDP (13 nucleotides deletion). Finally, it is also proposed that increased activity of the HPFH promoters may secondarily cause decreased expression of the delta- and beta-globin genes in cis possibly by competition between gamma- and beta-globin promoters for interaction with common regulatory elements.

4-Hydroxynonenal affects pRb/E2F pathway in HL-60 human leukemic cells

4-Hydroxynonenal (HNE), a highly reactive product of lipid peroxidation, has an antiproliferative effect in several tumor cell lines and provokes alteration of cell cycle progression in HL-60 cells. HNE down-regulates c-myc expression in K562, HL-60, and MEL cells. This prompted us to study the cascade of phenomena that, starting from the CKIs expression and the phosphorylation of pRb, arrives at the E2F binding to consensus sequence in the P2 promoter of the c-myc gene. Treatment of HL-60 cells with HNE (1 microM) causes a p53-independent increase of p21(WAF1/CIP1) expression, pRb dephosphorylation, a decrease of low molecular weight E2F complexes and an increase of high molecular weight E2F complexes bound to P2 c-myc promoter. E2F4 expression is reduced by HNE treatment as well as the amount of pRb/E2F4 complexes, whereas the amount of pRb/E2F1 complexes is increased. In conclusion, HNE can affect the pRb/E2F pathway by modifying the expression of several genes involved in the control of cell proliferation.

Direct demonstration of beta-globin mRNA in homozygous Ferrara betaO-thalassaemia patients.

In cases of β0- thalassaemia from Ferrara, Italy, the β-globin gene is transcribed into mRNA but no protein is synthesised. For these cases there is no hybridisation data suggesting a globin gene structural mutation. This again demonstrates the diverse molecular events which may cause this prevalent hereditary disease.

The same nuclear proteins bind the proximal CACCC box of the human β-globin promoter and a similar sequence in the enhancer

Using in vitro assays, we show that nuclear proteins related to the Sp1 and GT-1 factors bind to a CACCC box sequence in the human beta-globin enhancer, adjacent to binding sites for the erythroid-specific factor NFE1 and the ubiquitous factor CP1. The same proteins are known to bind to the proximal, but not to the distal, CACCC, box in the human beta-globin promoter. A C G mutation in the promoter CACCC box, known to cause beta-thalassemia, greatly decreases protein binding to the CACCC box; the same effect is obtained when this mutation is introduced into the enhancer CACCC box.

PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells

In human cells the length of telomeres depends on telomerase activity. This activity and the expression of the catalytic subunit of human telomerase reverse transcriptase (hTERT) is strongly up‐regulated in most human cancers. hTERT expression is regulated by different transcription factors, such as c‐Myc, Mad1 and Sp1. In this study, we demonstrated that 15d‐PG J2 and rosiglitazone (an endogenous and synthetic peroxisome proliferators activated receptor γ (PPARγ) ligand, respectively) inhibited hTERT expression and telomerase activity in CaCo‐2 colon cancer cells. Moreover, both ligands inhibited c‐Myc protein expression and its E‐box DNA binding activity. Additionally, Mad1 protein expression and its E‐box DNA binding activity were strongly increased by 15d‐PG J2 and, to a lesser extent, by rosiglitazone. Sp1 transcription factor expression and its GC‐box DNA binding activity were not affected by both PPARγ ligands. Results obtained by transient transfection of CaCo‐2 cells with pmaxFP‐Green‐PRL plasmid constructs containing the functional hTERT core promoter (including one E‐box and five GC‐boxes) and its E‐box deleted sequences, cloned upstream of the green fluorescent protein reporter gene, demonstrated that 15d‐PG J2, and with minor effectiveness, rosiglitazone, strongly reduced hTERT core promoter activity. E‐boxes for Myc/Mad/Max binding showed a higher activity than GC‐boxes for Sp1. By using GW9662, an antagonist of PPARγ, we demonstrated that the effects of 15d‐PG J2 are completely PPARγ independent, whereas the effects of rosiglitazone on hTERT expression seem to be partially PPARγ independent. The regulation of hTERT expression by 15d‐PG J2 and rosiglitazone, through the modulation of the Myc/Max/Mad1 network, may represent a new mechanism of action of these substances in inhibiting cell proliferation.

A frequent Aγ-persistence of fetal hemoglobin in northern Sardinia: its molecular basis and hematologic phenotype in heterozygotes and compound heterozygotes with β-thalassemia

SummaryA survey of hemoglobinopathies in northern Sardinia revealed a high frequency (0.3%) of carriers of a hematologic condition characterized by increased expression of fetal hemoglobin during adult life (hereditary persistence of fetal hemoglobin or HPFH). In spite of a normal hematologic phenotype, the heterozygous carriers for this condition display about 12% HbF, almost exclusively of the Aγ type; compound heterozygotes with β-thalassemia have 20%–26% HbF and run a very mild clinical course. The sequence analysis of the cloned Aγ gene linked to the HPFH determinant revealed the presence of a G→A substitution at position-117 of the Aγ- gene promoter; the same mutation occurs also in Greek HPFH, although associated with different restriction polymorphisms. Another hereditary condition characterized by increased HbF (α2Aγ2) level and a mild thalassemic phenotype in Sardinia is associated with the-196 C→T substitution in the Aγ-globin gene promoter (Sardinian δβ-thalassemia). Population studies using oligonucleotides complementary both to the-117 G→A and-196 C→T mutations and the corresponding normal sequences confirm the presence of these mutations only in HPFH and δβ-thalassemia chromosomes and exclude these changes being common DNA polymorphisms.

Isoelectric Focusing of Globin Chains for Antenatal Diagnosis of β°-Thalassemia

A recently developed isoelectric focusing technique for human globin chain separation has been applied to the antenatal diagnosis of β°-thalassemia in Sardinia. Results obtained with this method show a complete concordance with those obtained by the currently-in-use chromatographic separation of globin chains by carboxymethyl-cellulose. The ease with which several samples (up to 20) can be simultaneously processed and analyzed by a single operator and the very simple equipment required make this new method ideal for the antenatal diagnosis of β°-thalassemia and could encourage a more widespread use of prenatal diagnosis of thalassemias.