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.

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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.

Brief Reporthaematologica | 2011; 96 (5) 767 IntroductionPersistent expression of fetal hemoglobin (HbF) is of great clinical relevance given its role in the amelioration of the phenotype of beta-thalassemia and sickle cell anemia. Several studies have identified genes and genetic variants controlling HbF levels in adults (HBG1/HBG2, HBS1-MYB and BCL11A) able to improve the severity of the two major beta-hemoglobinopathies, beta thalassemia and sickle cell anemia. [1][2][3][4] Recently a nonsense mutation in the KLF1 gene, which ablates the DNA binding domain of this key erythroid transcriptional regulator, has been reported in a large Maltese family with hereditary persistence of HbF (HPFH). 5,6 Haploinsufficiency of KLF1 expression has been considered to be responsible for HPFH.In the Sardinian family described here, we found a marked increase of HbF only in compound heterozygotes for two KLF1 mutations and we did not confirm the KLF1 haploinsufficiency as a cause of HPFH. Moreover, we report, for the first time in humans, very high levels of zinc protoporphyrin associated with KLF1 mutations. Design and MethodsWe studied a Sardinian family with HPFH. Blood samples were obtained after informed consent. Hematologic and biochemical analyses were performed according to standard procedures. Zinc protoporphyrin in RBC was determined with ZPP hematofluorometer (AVIV Biomedical, Lakewood, NJ, USA) and blood protoporphyrin IX with a fluorometric method.Genomic DNA was obtained from peripheral blood by standard methods.Mutation analysis was performed by PCR amplification and DNA sequence analysis of the KLF1 gene using previously described primers.6 Genotyping of individual SNPs in the HBS1L-MYB (rs9399137) and BCL11A (rs11886868) loci was performed using Taqman genotyping assay (Applied Biosystem, Warrington, UK). Alpha globin and bilirubin UDP-glucuronosyltransferase (UGT1A1) gene genotyping was carried out as previously described. 7,8 The site directed mutation in K332Q in the KLF1 cDNA was obtained with the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA).Band shift, supershift, Western blots and transactivation analysisCompound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin ABSTRACT were performed as previously described. 9 The intensities of the KLF1 shifted bands were determined with the ImageQuant software after gel autoradiography on phosphoscreen and acquisition with PhosphoImager Storm 840 (GE Healthcare).The study was approved by the institutional review board of the hospital (ASL8 Ethics Commitee).

In this study we have defined the molecular basis and correlated the clinical phenotype with the α-globin genotype in a large series of patients of Sardinian descent with HbH disease. The most prevalent molecular defect was the deletion of 3 α-globin structural genes most commonly the (– –/–α^3.7) genotype (83.6%) and rarely the (– –/– α^4.2) genotype (1.4%), followed in decreasing order of incidence by the combination of deletion α°-thalassemia and initiation codon mutation of the α₂-gene (– –/α^NcoIα = 9.8%), deletion α°-thalassemia and pentanucleotide deletion of IVS-I of the α₂-globin gene, (– –/α^HphIα = 3.3%) deletion α°-thalassemia and initiation codon mutation of the α₁-gene (– –/αα^NcoI = 1.3%), a homozygous state for initiation codon mutation of the α₂-gene (α^Ncoα/α^NcoIα = 0.7%). Patients with the (– –/α^thalα) genotypes showed severer clinical and hematological features as compared to those with the (– –/–α) or those with the (– –/αα^thal) genotypes. The single patient with the (α^Ncoα/α^Ncoα) genotype had a clinical phenotype intermediate between HbH disease and the α-thalassemia carrier status. This heterogeneity depends on the fact that the α₂-globin gene produces 2-3 times α-globin chains than the α₁-gene and the single remaining α₁-like globin gene in the -α^{3 7} chromosome has a compensatory increase in the α-globin chain output. α-Globin gene mapping of HbH disease patients may be useful for predicting the clinical outcome and to improve genetic counselling.