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Introduction: Analysis of fetal DNA in at risk couples for thalassemia is performed from fetal trophoblast or amniotic fluid cells. Although these procedures are in common use, the main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity develops around 4 weeks of pregnancy within the extraembryonic mesoderm and contains embryonic erythroid precursor cells as a source of fetal DNA that can be used to perform invasive prenatal diagnosis., Methods: Celomatic fluids were obtained at 8 weeks of gestation in thirteen women with high-risk pregnancies. Twelve of these couples were at risk for Hb Lepore disease and β-thalassemia and one couple represented a rare case in which both parents were carriers of Hb Lepore Boston-Washington. Fetal cells were isolated by micromanipulator and nested polymerase chain reactions were performed., Results: The analysis was successfully performed in all examined cases. Two fetuses were found to have a compound heterozygosity for β-thalassemia and Hb Lepore Boston-Washington, three fetuses were found to be carriers of β-thalassemia, three fetuses of Hb Lepore, five were found without parental mutations. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA., Conclusion: Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in celomatic fluid and demonstrate for the first time that prenatal diagnosis of β-thalassemia and Hb Lepore may be feasible in an earlier time of pregnancy than other procedures., (© 2022 John Wiley & Sons Ltd.)

HBD: c.442T>C is a new mutation at the stop codon (TGA>CGA) of the δ-globin gene, which produces a new codon for arginine. This substitution causes a 51 nucleotides longer open reading frame determining the synthesis of a potential larger δ subunit, which is a probable target of mechanisms for the degradation of aberrant proteins as well as the defective synthesized mRNA molecules, and may also be rapidly degraded by a variety of RNA surveillance pathways. We identified this molecular defect in four patients: three women with a reduced HbA 2 level and a 37-year-old male showing the typical phenotype of an α-thalassemia (α-thal) carrier with reduced values of red cell indices and normal HbA 2 level (2.5%). The mutation on the δ-globin gene was found to have been coinherited with a β-globin gene defect leading to a normalized HbA 2 level. These data support the necessity of investigating these cases at a molecular level, particularly if the partner is also a β-thalassemia (β-thal) carrier. The present data emphasizes the importance of a careful evaluation of correlation between genotypes resulting from DNA analysis and phenotypes, especially in cases of atypical hematological parameters, in order to carry out an adequate diagnostic process finalized to appropriate genetic counseling.

A 59-year-old Italian woman came to our center for revaluation of a previous diagnosis of polycythemia vera. The patient presented with a lifelong history of polycythemia, no increase in white blood cells (WBCs) and platelets, and a negative bone marrow biopsy. Analysis of hemoglobin (Hb) fractions showed an abnormal fast moving Hb component. We aimed to determine if this variant was the cause of polycythemia in this patient. A complete blood count (CBC) was performed by an automated cell counter and Hb fractions were determined by high performance liquid chromatography (HPLC). Standard stability tests and oxygen affinity evaluation were also performed. Genomic DNA was extracted from peripheral blood leukocytes using the phenol chloroform method and the entire β-globin gene was analyzed by direct sequencing. At the hematological level, no anemia or hemolysis was observed but an abnormal Hb fraction was detected using cation exchange HPLC. Molecular analysis of the β-globin gene showed heterozygosity for an AAG > ACG substitution at codon 144, resulting in a Lys→Thr amino acid replacement. We demonstrated that this is a new Hb variant with increased oxygen affinity. Its altered physiology is caused by the reduction of 2,3-diphosphoglycerate (2,3-DPG) effects, due to an amino acid substitution in the central pocket near the C-terminal of the β chain. We called this new variant Hb San Cataldo for the native city of proband.

Purpose: Nearly 1183 different molecular defects of the globin genes leading to hemoglobin variants have been identified (http://globin.bx.psu.edu) over the past decades. The purpose of this study was to report three cases, never described in the literature, of co-inheritance of three β hemoglobin variants with other alterations in globin genes and to evaluate the clinical significance to conduct an appropriate genetic counseling., Patients and Methods: We report the molecular study performed in three probands and their families, sampling during the screening program conducted at the Laboratory for Molecular Prenatal Diagnosis of Hemoglobinopathies at Villa Sofia-Cervello Hospital in Palermo, Italy., Results: This work allowed us to describe the co-inheritance of three rare β hemoglobin variants with other alterations in globin genes: the β hemoglobin variant Hb Yaounde [β134(H12)Val>Ala], found for the first time in combination with ααα(anti3.7) arrangement, and the β hemoglobin variants Hb Görwihl [β5(A2)Pro>Ala] and Hb City of Hope [β69(E13)Gly>Ser], found both in association with β(0) -thalassemia., Conclusion: The present work emphasizes the importance of a careful evaluation of the hematological data, especially in cases of atypical hematological parameters, to carry out an adequate and complete molecular study and to formulate an appropriate genetic counseling for couples at risk., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Over the past two decades, a wide range of available methods for DNA analysis have allowed us to identify defects in globin genes associated with haemoglobin disorders and to correlate specific mutations with phenotypic expression. The purpose of this study was to evaluate the nature of three new nucleotide changes, mutation or single nucleotide polymorphism, found in the beta-globin gene, to conduct an appropriate genetic counselling., Patients and Methods: We report the molecular study performed in three probands and their families, sampling during the screening programme conducted at the Laboratory for Molecular Prenatal Diagnosis of Hemoglobinopathies at Villa Sofia-Cervello Hospital in Palermo, Italy., Results: This work allowed us to report three new nucleotide substitutions of the β-globin gene: a substitution of the nucleotide 16 in the CAP site area (HBB: c.-35 A>G), a substitution of the nucleotide 478 in the second intron (HBB: c.316-373) in association with β-haemoglobin variant Hb G Copenhagen (HBB:c.142G>A) and a substitution of the nucleotide 1656 within the 3' UTR (HBB: c.*+182 G>A) in association with the 1393-bp deletion (NG_000007.3:g.70060_71452del1393)., Conclusion: The present work emphasizes the importance of reporting the observed nucleotide changes to the Haemoglobin Variant Database, especially in the case of new or rare undefined mutations, to facilitate the determination of their phenotypic expression and the possible interactions with known molecular defects and to formulate an appropriate genetic counselling for couples at risk., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of this study is to update the incidence and the distribution of the globin gene defects causing β-thalassemia and abnormal hemoglobins in Sicily. The data derived from a total of 8875 beta-thalassemia alleles and 1330 variant hemoglobin chromosomes studied in Sicily from 1990 during a hemoglobinopathy control program. Fifty-four beta-globin gene defects were characterized, involving 30 different beta-thalassemia mutations and 24 variant hemoglobins. Eight of 30 β-thalassemia defects accounted for 95.11% of examined alleles while other beta-globin gene defects were found at lower frequencies (<1%). A consistent number (24) of variant hemoglobins were identified of whom Hb S was the most represented (72.1%). Our data underline the heterogeneity of the beta-globin gene defects in the Sicily. The enormous progress in the technique for β-globin gene analysis permitted to characterize 99.93% of mutated alleles and it has made a first trimester prenatal diagnosis program possible in our region in all cases with a great improvement in thalassemia management. The origin of the large spectrum of mutations is discussed taking in consideration the history of the island., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report a new hemoglobin (Hb) variant, Hb Marineo [beta70(E14)Ala --> Val], found in three generations of a family from West Sicily. The mutation is due to a GCC --> GTC substitution at codon 70 of the beta-globin gene. To date, three mutations at codon 70 of the beta-globin gene have been described, presenting with hemolytic anemia. In our case, no anemia or other alteration of hematological indices were found. Cation exchange high performance liquid chromatography (HPLC) showed a peak in the P2 window (VARIANT I), while a peak was detected by VARIANT II HPLC in the P3 window. Reversed phase HPLC analysis showed an abnormal chain amounting to about 40% of the total beta chains.