F. Mavilio

Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac----liver transition.

Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS----L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts----macrocytes) and globin expression (zeta----alpha, epsilon----gamma).

Heterocellular hereditary persistence of fetal hemoglobin (HPFH). Molecular mechanisms of abnormal γ-gene expression in association with β thalassemia and linkage relationship with the β-globin gene cluster

SummaryWe report a study of four families of Italian origin in which heterocellular HPFH is inherited linked to β thalassemia over two or three generations. The HPFH + β thalassemia carriers showed thalassemic blood pictures and elevated HbF and F-cell number without increase in the HbF/F-cell content. Association of this gene complex with a second β thalassemia trait gives rise to a mild clinical picture characterized by 9–12 g/dl of mainly HbF in peripheral blood and no transfusion requirement. In two families, independent segregation of the HPFH or β-thal trait was observed, and in one case the study of the DNA polymorphisms within the γδβ gene cluster indicated that the HPFH mutation lies outside that DNA region. In one family the coexistence of a polymorphic variant of the Aγ chain (the AγT chain) allowed us to demonstrate that the increased γ chain synthesis caused by the heterocellular HPFH determinant is directed by both chromosomes.

Haemoglobin Lepore trait: haematological and structural studies on the Italian population.

Summary. Haematological data on 59 heterozygotes for haemoglobin (Hb) Lepore and 10 double heterozygotes for Hb Lepore and β thalassaemia from 36 Italian families are reported. The red cell indices are defined and compared with those of groups of non‐thalassaemic and β thalassaemic subjects of comparable number, age and sex distribution. The relative level of each haemoglobin fraction and the absolute production of single polypeptide chains are calculated in order to compare the expression of the non‐α chain genes in Hb Lepore trait and β thalassaemia. Structural studies demonstrate that the haemoglobin Lepore is of the Boston type (δ87β116) in all subjects, confirming that this type of fusion variant is probably the only one which occurs in Mediterranean populations. The distribution and incidence of the Lepore haemoglobinopathy are discussed.

A new abnormal human hemoglobin: Hb prato (α231 (B12) Arg→Ser β2)

Abstract An abnormal human hemoglobin was found in a hemolysate from a 5-year-old healthy child living in Prato (Tuscany, Italy). Structural studies demonstrated a previously unreported amino acid substitution, α31 (B12) Arg → Ser (this is an α 1β1 contact). The new variant has been named Hb Prato. It was unstable in isopropanol and heat-denaturation tests, but has normal functional properties, with respect to whole blood studies. Family studies indicated that the variant had been inherited from the mother, a 39-year-old woman of Sicilian extraction. Hb Prato occurs at 20 and 28% in hemolysates from the boy and woman, respectively.

Molecular heterogeneity of beta thalassaemia in the Italian population

Summary Fifty‐one subjects originating from Southern Italy and affected by Cooleys anaemia have been studied in order to define the degree of heterogeneity of β thalassaemia mutations in this high incidence area. Restriction endonuclease mapping has been carried out on genomic DNA by the Southern blot technique both to exclude the existence of gross deletions or rearrangements and to establish the relative frequency of four polymorphic restriction sites (i.e. Gγ and Aγ Hind III, β Ava II and β Bam HI) within the γδβ gene region. In 28 subjects unequivocal linkage of the four polymorphic sites has been determined leading to the identification of seven different chromosome haplotypes, six of which had previously been reported associated with specific β0 and β+ thalassaemia mutations. Globin chain synthesis studies on peripheral blood reticulocytes indicated that subjects carrying the same genotype may behave differently as far as the β chain production is concerned relative to both the a and the non‐α chains. Thus, β thalassaemia turns out to be quite heterogeneous even in this limited geographical area. β+ mutations appear to be predominant, particularly those affecting nuclear precursor RNA splicing to mature β globin mRNA.

Hemoglobin bologna (α2β2 61 (E5) Lys → Met) An abnormal human hemoglobin wlth low oxygen affinity

Abstract An abnormal human hemoglobin was found in association with β-thalassemia in a hemolysate from an 11-year-old healthy child living in Bologna (northern) Italy). Structural studies demonstrated a previously unreported amino acid substitution, β 61 (E5) Lys → Met (this is an external residue). The new variant has been named Hb Bologna, and is characterized by a reduced oxygen affinity. Family studies indicated that the variant had been inherited from the father, a 41-year-old male of Southern Italian origin. Also, a brother of the propositus was found to be an abnormal Hb carrier.

Post-translational control of human hemoglobin synthesis The role of the differential affinity between globin chains in the control of mutated globin gene expression

The interactions between beta-thalassemia and the human hemoglobin (Hb) alpha-chain variants, Hb Hasharon, Hb O Idonesia and Hb J Paris, and between alpha-thalassemia and the beta-chain variants, Hb S, Hb C and Hb G San José, which are characterized by preferential decrease of the abnormal Hb level in peripheral bloods, have been studied. Both biosynthesis studies in reticulocytes and determination of the relative affinity of abnormal chains for normal complementary chains by in vivo recombination experiments, involving globin chains previously isolated in their native form, have been carried out in order to provide insights on the molecular events following the synthesis of the mutant chains under conditions of complementary chain deficiency. Furthermore, we have measured the relative affinity for complementary chain of beta D Los Angeles- and alpha J Rovigo-chains, the level of which does not decay in thalassemic carriers, and of alpha Legnano- and beta Osu Christiansborg-chains, which have not yet been observed in association with thalassemias. Our experiments indicated that the differential affinity for beta-chains is not always the major post-translational control mechanism which regulates the level of certain alpha-chain variants in beta-thalassemic heterozygotes, and that preferential removal of abnormal chains by proteolytic enzymes is likely to play an important role in most cases. On the other hand, the low affinity of certain variant beta-chains for alpha-chains may offer an explanation for the low level of certain beta-chain variants in peripheral blood of non-thalassemic carriers, as well as to their decrease under conditions of relative alpha-chain deficiency (alpha-thalassemias).

A new human hemoglobin variant: Hb Bari (α2 45 (CD3) His → Gln β2)

Abstract An α-chain variant hemoglobin was found in the hemolysate of a 21-year-old healthy male living in Bari (Puglia, Italy). Structural studies demonstrated a previously unreported amino acid substitution, α2 45 (CD3) His → Gln β2, involving a distal heme contact. The new variant has been named Hb Bari. Its electrophoretic behavior was the same as for Hb A; it was stable to both isopropanol and heat denaturation and exhibited normal functional properties, with respect to whole blood and stripped hemolysate studies. The level of Hb Bari was about 20% in the observed carrier. No relative was available for further investigations.

Hemoglobin Gavello - α2β2 47(CD6) Asp → Gly a new Hemoglobin Variant from Polesine (Italy)

During a survey for abnormal hemoglobins in Polesine (a region north of the Po river, where betathalassemia is very frequent) a slow moving variant was noted in a 79-yr-old woman living in Gavello, a small town in the province of Rovigo. Structural studies demonstrated a previously undescribed amino acid substitution, 647 Asp a Gly. This new variant has been named Hb Gavello.

Expression of transferrin receptors: differential regulatory mechanisms in monocytes-macrophages versus other hemopoietic cells.

Interaction of a cell membrane receptor with its ligand induces either activation of a specific biological process or cellular uptake of an essential nutrient. On this basis, receptors have been classified in two categories:’ class I receptors transmit a specific piece of information (e.g., epidermal and platelet-derived growth factor receptors modulate the growth of a variety of cell types); class I1 receptors interact with and internalize glycoproteins carrying essential metabolic factors (e.g., low-densitylipoproteins (LDL) and transferrin (Trf) receptors allow respectively the uptake of cholesterol and iron). Upon exposure to ligand, class I receptors are rapidly downregulated. In contrast, binding of ligand with class I1 receptors does not usually lead to modulation of their number. A paradigmatic class I1 receptor is the human receptor for LDL.’ Incubation of cells with cholesterol-saturated LDL results in a decrease in the number of receptors. Conversely, incubation in the absence of LDL causes a rise in their number. Both phenomena are mediated by modulation of receptor synthesis, which is in turn controlled by the intracellular concentration of free cholesterol.2 Recent studies suggest that the Trf receptor may similarly represent a typical class I1 receptor: the level of intracellular iron apparently modulates the rate of Trf receptor synthesis,’ thus resembling the regulation of LDL receptors via cholesterol. All types of cells require iron to sustain essential metabolic pathways. Additionally, actively dividing cells need iron for their growth, presumably because the metal is