Liliana Maccioni

Origin, Diffusion, and Differentiation of Y-Chromosome Haplogroups E and J: Inferences on the Neolithization of Europe and Later Migratory Events in the Mediterranean Area

The phylogeography of Y-chromosome haplogroups E (Hg E) and J (Hg J) was investigated in >2400 subjects from 29 populations, mainly from Europe and the Mediterranean area but also from Africa and Asia. The observed 501 Hg E and 445 Hg J samples were subtyped using 36 binary markers and eight microsatellite loci. Spatial patterns reveal that (1). the two sister clades, J-M267 and J-M172, are distributed differentially within the Near East, North Africa, and Europe; (2). J-M267 was spread by two temporally distinct migratory episodes, the most recent one probably associated with the diffusion of Arab people; (3). E-M81 is typical of Berbers, and its presence in Iberia and Sicily is due to recent gene flow from North Africa; (4). J-M172(xM12) distribution is consistent with a Levantine/Anatolian dispersal route to southeastern Europe and may reflect the spread of Anatolian farmers; and (5). E-M78 (for which microsatellite data suggest an eastern African origin) and, to a lesser extent, J-M12(M102) lineages would trace the subsequent diffusion of people from the southern Balkans to the west. A 7%-22% contribution of Y chromosomes from Greece to southern Italy was estimated by admixture analysis.

In Vitro Mass Production of Human Erythroid Cells from the Blood of Normal Donors and of Thalassemic Patients

We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (10(5) cells/mL) or light-density (10(6) cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10(-6) M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated approximately 1.2 x 10(7) erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45(low)/glycophorin (GPA)(neg)/CD71(1ow) cells at day 7, 50-60\% of which became CD45(neg)/GPA+/CD71high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (g90% benzidine(pos) and CD45(neg)/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.

Compound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin

The persistence of high fetal hemoglobin level in adults may ameliorate the clinical phenotype of beta-thalassemia and sickle cell anemia. Several genetic variants responsible for hereditary persistence of fetal hemoglobin, linked and not linked to the beta globin gene cluster, have been identified in patients and in normal individuals. Monoallelic loss of KLF1, a gene with a key role in erythropoiesis, has been recently reported to be responsible for persistence of high levels of fetal hemoglobin. In a Sardinian family, high levels of HbF (22.1–30.9%) were present only in compound heterozygotes for the S270X nonsense and K332Q missense mutations, while the isolated S270X nonsense (haploinsufficiency) or K332Q missense mutation were associated with normal HbF levels (<1.5%). Functionally, the K332Q Klf1 mutation impairs binding to the BCl11A gene and activation of the γ- and β-globin promoters. Moreover, we report for the first time the association of KLF1 mutations with very high levels of zinc protoporphyrin.

HbH Disease in Sardinia: Molecular, Hematological and Clinical Aspects

In this study we have defined the molecular basis and correlated the clinical phenotype with the alpha-globin genotype in a large series of patients of Sardinian descent with HbH disease. The most prevalent molecular defect was the deletion of 3 alpha-globin structural genes most commonly the (--/-alpha 3.7) genotype (83.6%) and rarely the (--/-alpha 4.2) genotype (1.4%), followed in decreasing order of incidence by the combination of deletion alpha zero-thalassemia and initiation codon mutation of the alpha 2-gene (--/alpha NcoI alpha = 9.8%), deletion alpha zero-thalassemia and pentanucleotide deletion of IVS-I of the alpha 2-globin gene, (--/alpha HphI alpha = 3.3%) deletion alpha zero-thalassemia and initiation codon mutation of the alpha 1-gene (--/alpha alpha NcoI = 1.3%), a homozygous state for initiation codon mutation of the alpha 2-gene (alpha Nco alpha/alpha NcoI alpha = 0.7%). Patients with the (--/alpha thal alpha) genotypes showed severer clinical and hematological features as compared to those with the (--/-alpha) or those with the (--/alpha alpha thal) genotypes. The single patient with the (alpha Nco alpha/alpha Nco alpha) genotype had a clinical phenotype intermediate between HbH disease and the alpha-thalassemia carrier status. This heterogeneity depends on the fact that the alpha 2-globin gene produces 2-3 times alpha-globin chains than the alpha 1-gene and the single remaining alpha 1-like globin gene in the -alpha 3.7 chromosome has a compensatory increase in the alpha-globin chain output. alpha-Globin gene mapping of HbH disease patients may be useful for predicting the clinical outcome and to improve genetic counseling.

α-Thalassemia carrier identification by DNA analysis in the screening for thalassemia

Differentiation between heterozygous α‐thalassemia and several phenotypically resembling alleles at the β‐globin gene cluster such as coinherited δ‐ and β‐thalassemia or γδβ‐thalassemia is a critical step in genetic counseling. In this paper we report our experience in the identification of the α‐thalassemia carrier state using polymerase chain reaction (PCR)‐based methods, and the feasibility and simplification of screening for thalassemia using this approach. α‐Globin genotype was determined by PCR‐based method in 526 adult subjects with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal hemoglobin A2 and F, and normal serum iron. To verify the reliability of the protocol used, in 68 of these subjects we performed globin chain synthesis analysis and in 101 we determined α‐globin genotype by Southern blot analysis. Five hundred twenty‐one (99%) of 526 subjects examined were identified as carriers of one or two α‐thalassemia alleles. The identification of the α‐thalassemia carrier state may be fast and accurate by PCR‐based method, avoiding other cumbersome and expensive methods such as globin chain synthesis and Southern blot analysis. Am. J. Hematol. 59:273–278, 1998.

Somatic deletion of the normal beta-globin gene leading to thalassaemia intermedia in heterozygous beta-thalassaemic patients.

Two β‐thalassaemia patients, whose constitutive genotype was β39C/β39C→T, had the clinical phenotype β‐thalassaemia intermedia. Analysis of leucocyte DNA showed the presence of the mutated β39C→T‐gene exclusively, while the normal β39C‐gene was also present in reticulocyte RNA. Deletional analysis of chromosome 11p15.5 on leucocyte DNA showed large deletions including the β‐globin gene. Two populations of erythroid progenitors, one heterozygous and the other hemizygous for the β39C→T mutation, were demonstrated in one case. This confirms that, in heterozygous individuals, β‐thalassaemia intermedia may be caused by inactivation of the β‐locus in trans as a result of chromosome 11p15.5 deletions in a subpopulation of haematopoietic cells.

β° Thalassemia Trait in Sardinia

The red cell indices and results of globin chain synthesis in peripheral blood of obligate β° thalassemia (β° thal) carriers (parents of hamozygous β° thal children) and β thalassemia (β thal) carriers identified during mass screening are reported. Red cell indices were similar in obligate β° carriers and in carriers diagnosed during mass screening. However there was a higher incidence of anemia in female obligate β° thal carriers. In Sardinia the β° thal carrier showed the usual hematological characteristics of the high Hb A2 β thal carrier with microcytosis, hypochromia, reduced osmotic fragility: Hb F > 1% was found in 30% of the carriers. With MCV, MCH, osmotic fragility test (OFT) and Shine and Lal discriminant function we found 3.5%, 1.5%, 3.5% and 4.0% respectively false negatives in carrier identification. A part from one subject, all obligate carriers had elevated Hb A2 levels. The α/β ratio in obligate carriers (mean±SD) was 1.83±0.26 (N=30).

Evaluation of an automatic HPLC analyser for thalassemia and haemoglobin variants screening

In this paper the authors report the evolution of a new automatic HPLC analyser for screening haemoglobinopathies. HbA2 and F determinations are accurate and reproducible. The analysis time is short (6.5 min) and there is a good separation between the HbA2 values of β-thalassemia carriers from normals and α-thalassemia carriers, with no overlap between these groups. In addition, the system is also able to detect and quantitate most of the haemoglobin variants, particularly those (HbS, HbC, HbE and Hb Lepore) able to interact with β-thalassemia and could make haemoglobin electrophoresis unnecessary in all samples. The ease of operation and the limited technical work make this system especially suitable for laboratories with a high workload and allow the cost of screening to be reduced.

Delayed fetal hemoglobin switching in subjects with KLF1 gene mutation

Variations at the KLF1 gene have been associated with a series of human erythroid phenotypes including the In-(Lu) phenotype, hereditary persistence of fetal hemoglobin, congenital dyserythropoietic anemia, borderline HbA(2) and increased red blood cell protoporphyrin. Natural mutations have shown that KLF1 regulates gamma globin gene expression and its role in the switching from fetal to adult globin expression has been suggested by experimental studies. In this paper we report that subjects with S270X KLF1 mutations show a decrease of HbF levels with increasing age, supporting in vivo the role of KLF1 in hemoglobin switching in humans.

Heterozygous β‐thalassemia with thalassemia intermedia phenotype

In this study we investigated the molecular bases of the β‐thalassemia intermedia phenotype in six patients belonging to two unrelated families of Sardinian descent. Sequence analysis of the β globin gene from these patients detected, as the sole abnormality, the heterozygosity for the codon 39 nonsense mutation. The Aγ and Gγ promoters as well as the HS2 and HS3 core sequences of the β globin LCR from these patients, did not show any non‐polymorphic nucleotide variation from the consensus sequence. One of the parents was heterozygous for codon 39 nonsense mutation but showed the β‐thalassemia carrier phenotype; the other was hematologically normal and had an entirely normal β globin gene sequence. In both families, other members showed the typical hematological phenotype, clinically silent, of heterozygous β thalassemia. To explain the thalassemia intermedia phenotype, we postulated the presence of an unknown molecular defect interacting with the β globin gene mutation. Haplotype analysis excluded that this postulated defect lies in the β globin gene cluster. Am. J. Hematol. 57:43–47, 1998.