R. Testa

Clinical, Hematological, and Molecular Features in Sicilians with Sickle Cell Disease

We report the clinical, hematological, and molecular findings observed in 32 Sicilian patients with sickle cell disease. None of our patients received regular blood transfusions and careful infectious disease prophylaxis was carried out for all. Haplotyping of beta S chromosomes was performed in all patients; all were homozygous for haplotype #19 (Benin). Gene mapping excluded the presence of an alpha-thalassemia in 13 of our patients; none of the relatives showed any evidence of the presence of alpha-thalassemia. Hb F levels were 11.8 +/- 5.9% with G gamma representing 39.6 +/- 3.6% of total gamma chain. Hb F levels were higher in females than in males (12.5 +/- 5.9% versus 9.7 +/- 6.5%) but the difference was not statistically significant. All patients, regardless of age and sex, were anemic with normal mean corpuscular hemoglobin concentration, high mean corpuscular volume and mean corpuscular hemoglobin, and mild reticulocytosis. Analysis of clinical manifestations suggests that our patients have a disease of moderate severity.

Hb Bronte or α93(FG5)Val→Gly: A New Unstable Variant of the α2‐Globin Gene, Associated with a Mild α+‐Thalassemia Phenotype

We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides −181 to +894 (α1) and to +884 (α2) revealed a GTG→GGG substitution at codon 93 of the α2‐globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3–0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3–4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the α chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the α chains was much higher than that of the β‐globin chains, and the α/β biosynthetic ratio in the mother and proband was of the β‐thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H‐specific activity of the peak containing normal and variant α chains was not linear and was much higher than that of β chains; moreover, the α/β biosynthetic ratio varied during the 2 hours incubation.

Presence of hemoglobinopathies in Sicily: a historic perspective.

Sicily, at the center of the Mediterranean, has been the meeting place of Eastern and Western civilizations, and in the Sicilian population the presence of many different alterations in the globin gene clusters can surely be considered testimony of past colonizations. From 1975 to 1994, 100,000 Sicilian subjects were screened by us to monitor the presence of hemoglobin (Hb) structural variants. In this paper we present the data gathered, emphasizing the high incidence (2.5%) of carriers of at least one abnormal Hb, and the great heterogeneity of globin molecular defects on the island. Twenty-six different mutations were identified: the most common occur in the beta-globin gene (beta(S), beta(C), deltabeta(Lepore), beta(G-San José), beta(O-Arab), but also quite frequent is the mutated allele alpha(J-Oxford). The chromosome haplotypes associated with some of them were characterized. Two uncommon Hbs, Copenhagen and D Punjab, and some 18 rare variants complete the wide spectrum of structural alterations of globin genes in Sicily. We think they are de novo mutations prevalently. It is not possible to exclude that the presence of a few of them is related to migratory phenomena, particularly from North Africa and East Asia. Numerous thalassemic alleles complete the picture of globin gene mutations in Silicy.

Comparative Approach to the Evaluation of Hemoglobin A2 by Two Different Methods: High-Performance Liquid Chromatography and DE-52 Microchromatography

Hb A2 was determined in 477 subjects: 77 were affected by iron deficiency anemia, 172 were carriers of beta-thalassemia trait and 228 were normal controls. Hb A2 was determined by both DE-52 microchromatography and high-performance liquid chromatography (HPLC). The analysis of the data by linear regression demonstrated that the methods furnish overlapping results. Our findings show that HPLC is a rapid and easily reproduced method which allows a quantitative and qualitative discrimination of the various Hb fractions, making it a valid tool in screening programs for hemoglobinopathies.

Association of Hb S/Hb lepore and δβ-thalassemia/Hb lepore in Sicilian patients: Review of the presence of Hb lepore in Sicily

Abstract: The hemoglobin (Hb) lepore‐Boston is a β‐globin structural variant, produced in a reduced amount and formed from the fusion of N‐terminus δ‐(residues 1–87) and C‐terminus β‐chains (residues 116–146). This type of fusion protein is quite common in Southern Italy (Campania, Calabria, and Sicily). We report here the hematological and hemoglobin data on 96 unrelated Sicilians with Hb lepore trait. Particularly interesting are the subjects where Hb lepore occurs with Hb S or Sicilian type δβ‐thalassemia. In these individuals, striking features are clinical variability and different hematological pictures. These observations underscore the importance of thalassemia screening in these geographic areas, such as Southern Italy, principally Sicily, where the mutations in globin gene clusters are especially prevalent. Moreover, as from the second half of the last century, owing to high migratory flux from Sicily to Northern Europe, North and South America, and Australia, the Hb lepore, as well as other hemoglobin variants, have become prevalent, making the identification of the heterozygotes a problem of general interest.

Relative Levels of α-, β-, and γ-mRNA from Patients with Severe and Intermediate β-Thalassemia Major

We have determined the relative quantities of γ- and β-mRNAs and the α/β-mRNA ratios in 37 patients with β-thalassemia major with specific genotypes, namely 8 with a homozygosity for codon (CD) 39 (C→

HbA2-Partinico or δ(A2)Pro→Thr, a new genetic variation in the δ-globin gene in cis to the β+ thal IVS-I-110 G>A, and the heterogeneity of δ-globin alleles in double heterozygotes for β- and δ-globin gene defects

The study of the alleles of the δ-globin gene is relevant to the prevention of β-thalassemia homozygosis; in fact, the increase of the HbA2 is an invaluable hematological marker of the β-thalassemia heterozygosis and the double heterozygosis for alleles of δ- and β-globin genes can cause the decrease of the HbA2 up to normal or borderline values. We carried out the characterization of alleles of the δ- and β-globin genes, restriction fragment length polymorphism (RFLP) haplotype background, and hematologic phenotype in 23 double heterozygotes belonging to 18 unrelated families. A wide heterogeneity of the δ-globin alleles was detected; seven known alleles in trans to the β-globin gene defects were revealed in 17 out of 18 families, while a new allele in cis to a β-thalassemia allele was detected in one family. Moreover, the relative frequency of the δ-mutants was quite different from that found among heterozygotes. The new allele δ-cod 5 CCT>ACT, in cis to the allele β+ thal IVS-I-110 G>A, was found in five carriers of a Sicilian family. The new variant δ5(A2)Pro→Thr, named HbA2-Partinico upon the origin of the family, was detected with high-performance liquid chromatography; it overlapped the HbA2 peak which was partially split. The double in cis heterozygotes had increased percentage of normal and variant HbA2 of comparable size. The variant originated most likely from a new mutational event because it was associated with RFLP haplotype I, commonly found with the β+ thal IVS-I-110 G>A, even if crossing over or gene conversion cannot be excluded.

Evidence for the Single Origin of Hb G-San Jose in Sicily

Hb G-San Jose [alpha 2 beta 2 7(A4)Glu----Gly] was detected in four families and three unrelated individuals from Eastern Sicily. Polymorphic restriction sites within the beta-globin gene cluster bearing the mutation were characterized. A complete association of beta-G-San Jose alleles with Mediterranean haplotype IV was found in the families examined and the same haplotype was also present in the unrelated individuals. These findings support the hypothesis of an unicentric origin of the beta-G-San Jose mutation which may have arisen in Eastern Sicily.

Unusual combination of genetic defects in a Sicilian family: β-thalassaemia, haemoglobin Lepore Boston-Washington and heterocellular hereditary persistence of fetal haemoglobin

Summary. This paper reports a Sicilian family in which β‐thalassaemia, haemoglobin Lepore Boston‐Washington and heterotocellular hereditary persistence of fetal haemoglobin (HPFH) were present in various combinations. The most interesting combination was that of Hb Lepore and heterocellular HPFH, which has not been previously reported. This subject was clinically normal, with the haematological picture of Hb Lepore trait and an unusually high level of Hb F due to an increased number of circulating F cells.

Hb G-SAN JOSÈ VARIANT LEVELS CORRELATE WITH α-THALASSEMIA GENOTYPES

Hb G-San Josè or β7(A4)Glu → Gly has been reported in Southern Italian or Mexican families. We have studied four families from Sicily and Campania, Southern Italy. In six carriers, the hemoglobin variant level ranged from 32 to 38%. In four double heterozygotes for Hb G-San Josè and α-thalassemia the variant level showed a strong correlation with the α-thalassemia genotype. In fact, the variant level was 15% when interacting with the −(α)20.5/αα, 19.6% with the αα/αPoly Aα, and 24.8% with αα/α−5 ntα genotypes. In two double heterozygotes for Hb G-San Josè and β+-IVS-I-6 (T → C) the hemoglobin variant level was 67%. These data show that the reduced synthesis of α chains causes drastic reduction of probability to form Hb G-San Josè in favor of the formation of Hb A. Moreover, this reduction, (i) correlates with the type of α-thalassemia genotype and with the degree of the α chain deficiency, and (ii) is, most probably, more marked than the degree of α chain reduction. The minor affinity of the β chain variant for the α chains associated with the reduced synthesis of the α chains is probably the principal cause of the variant hemoglobin reduction. Moreover, the rapid removal of the abnormal chains by proteolytic enzymes must have an essential role in order to reduce the chain variant pool. These conclusions are in agreement with the results obtained in reticulocyte and in vitro recombination experiments.