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Background: Turner syndrome is a rare genetic condition in which a female is partly or completely missing an X chromosome. Signs and symptoms vary among those affected. In fetuses that survive at birth and without congenital malformations, the prognosis is usually positive, but it has high lethality in utero, especially in the first trimester of pregnancy. Methods: We report a case of monosomy X detected during a prenatal diagnosis for beta thalassemia on coelomic fluid (CF) at the VIII week of gestation. Beta globin gene analysis, whole genome amplification (WGA), quantitative fluorescent PCR and array comparative genomic hybridization (array-CGH) were performed on DNA extracted from CF. Results: A monoallelic pattern of all Short Tandem Repeats mapped on the X chromosome was found and array-CGH performed on WGA from a few fetal erythroblasts confirmed monosomy X. Conclusion: This report underlines the importance of an early prenatal diagnosis and the countless potentialities of array-CGH that could make definition of molecular karyotype possible from a few fetal cells, unlike conventional cytogenetic techniques that require a greater cellular content. This is the first report of a molecular karyotype obtained from two cells selected by micromanipulation of CF and defined at such an early gestational age. [ABSTRACT FROM AUTHOR]

The procedures commonly used for prenatal diagnosis (PND) of thalassemia are villocentesis or amniocentesis, respectively, at the 11th and 16th weeks of gestation. Their main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity is accessible between the 7th and 9th weeks of gestation and it has been demonstrated that it contains embryonic erythroid precursor cells as a source of fetal DNA for earlier invasive PND of thalassemia and other monogenic diseases. In this study, we report the use of celomatic fluids obtained from nine women with high-risk pregnancies for Sicilian (δβ)0-thalassemia [(δβ)0-thal] deletion (NG_000007.3: g.64336_77738del13403) and β-thalassemia (β-thal). Fetal cells were isolated by a micromanipulator, and nested polymerase chain reaction (PCR) and short tandem repeats (STRs) analysis were performed. Prenatal diagnosis was successfully performed in all examined cases. One fetus was a compound heterozygote for (δβ)0- and β-thal, three fetuses were found to be carriers of β-thal, four fetuses carriers of a Sicilian δβ deletion, and one fetus without parental mutations. Accidentally, a rare case of paternal triploidy was observed. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in the celomatic fluid and demonstrate, for the first time, that PND of Sicilian (δβ)0-thal and β-thal is feasible at an earlier time in pregnancy than other procedures. [ABSTRACT FROM AUTHOR]

Objectives: We performed molecular analysis to evaluate clinical implications of a rare nucleotide change, particularly when co-inherited with other known mutations in the globin clusters, in order to conduct an appropriate genetic counselling. Methods: Complete blood cell counts and high-performance liquid chromatography were the routine first level analysis for patients referred to our Hospital Center in Palermo to undergo the screening test for haemoglobinopathies. Sequencing analysis was the selected method for the phenotypic characterization, especially in case of new or very rare mutations in globin genes. Results: We report data of a rare single nucleotide variation at position −56 relative to transcription initiation site (NM_000518.4(HBB):c.−106G > C), identified in ten patients of Italian origin during the screening programme of the ‘Sicilian population’. It was found in simple heterozygosity (n = 8), in association with beta haemoglobin variant Hb S (n = 1) and in heterozygosity with beta-thalassaemic allele IVS-I-1 G->A [(HBB):c.92 + 1G > A] and ααα anti3.7 rearrangement (n = 1). Discussion: Heterozygous subjects for this substitution showed normal haematological and electrophoretic features. Heterozygotes for this mutation and other defect in globin genes showed the classical phenotype of a healthy carrier, therefore it can be considered a benign variant that does not alter the production and function of haemoglobin. Conclusion: This is another example of rare or new nucleotide variations whose identification and characterization is crucial in order to carry out appropriate genetic counselling to a potential risk couple. [ABSTRACT FROM AUTHOR]

A large number of methods for DNA analysis are available to identify defects in globin genes associated with hemoglobin (Hb) disorders. In this study, we report a rare nucleotide (nt) substitution on the β-globin gene, nt 781 in the second intron [IVS-II-781 (C > G);HBB: c.316-70C > G], identified in four patients. This nt substitution was previously described only as a personal communication to the HbVar database and indicated as a β0or β+mutation. The purpose of this study was to evaluate the clinical implication of this nt change, particularly when coinherited with severe β-thalassemia (β-thal), in order to be able to conduct appropriate genetic counseling. Genetic studies were performed on two subjects, one carried Hb S [β6(A3)Glu→Val;HBB: c.20A > T], and the other carried IVS-I-110 (G > A) (HBB: c.93-21G > A). All these subjects showed this new β nt substitution in association with Hb A2' (or Hb B2) [δ16(A13)Gly→Arg;HBD: c.49G > C]. Another 16 samples, carrying the same δ variant as the probands, were processed by β-globin gene sequencing in order to better understand the correlation between this Hb variant and the rare nt substitution reported in this study. The present investigation emphasizes the importance of sharing the observed nt changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression, the possible interactions with known molecular defects and to formulate appropriate genetic counseling for at-risk couples. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: To evaluate the extent of interlaboratory variation and accuracy in hemoglobin A2 (HbA2) assays, a pilot study of external quality assessment was organized among 48 Italian laboratories routinely measuring HbA2. As part of the study, a survey was also performed by sending a questionnaire concerning some important analytical aspects related to the determination of HbA2. Methods: The trial specimens consisted of three whole blood samples (A, B and C) with normal, pathological and borderline HbA2 content, respectively. All laboratories used HPLC analyzers from the same manufacturer (Bio-Rad Laboratories). Results: Normal and pathological samples were clearly differentiated by all laboratories, while data for the borderline sample partially overlapped those for the other samples. The overall interlaboratory coefficient of variation was 8.0%, 6.0% and 7.9% for samples with low, high and intermediate HbA2 levels, respectively. To assign HbA2 target values to the samples, the median of the laboratory group was used. The accuracy of HbA2 results was evaluated on the basis of allowable total error. The proportion of laboratories reporting unacceptable results was 31.9% (15 out of 47) for sample A, 17.0% (8 out of 47) for sample B, and 31.9% (15 out of 47) for sample C. No abnormalities in the chromatographic separation pattern were reported by any of the laboratories. Conclusions: We conclude that quality in the measurement of HbA2 should be improved. Clin Chem Lab Med 2007;45:88–92. [ABSTRACT FROM AUTHOR]

The procedures commonly used for prenatal diagnosis (PND) of thalassemia are villocentesis or amniocentesis, respectively, at the 11th and 16th weeks of gestation. Their main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity is accessible between the 7th and 9th weeks of gestation and it has been demonstrated that it contains embryonic erythroid precursor cells as a source of fetal DNA for earlier invasive PND of thalassemia and other monogenic diseases. In this study, we report the use of celomatic fluids obtained from nine women with high-risk pregnancies for Sicilian (δβ) 0 -thalassemia [(δβ) 0 -thal] deletion (NG_000007.3: g.64336_77738del13403) and β-thalassemia (β-thal). Fetal cells were isolated by a micromanipulator, and nested polymerase chain reaction (PCR) and short tandem repeats (STRs) analysis were performed. Prenatal diagnosis was successfully performed in all examined cases. One fetus was a compound heterozygote for (δβ) 0 - and β-thal, three fetuses were found to be carriers of β-thal, four fetuses carriers of a Sicilian δβ deletion, and one fetus without parental mutations. Accidentally, a rare case of paternal triploidy was observed. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in the celomatic fluid and demonstrate, for the first time, that PND of Sicilian (δβ) 0 -thal and β-thal is feasible at an earlier time in pregnancy than other procedures.

Objectives: We performed molecular analysis to evaluate clinical implications of a rare nucleotide change, particularly when co-inherited with other known mutations in the globin clusters, in order to conduct an appropriate genetic counselling., Methods: Complete blood cell counts and high-performance liquid chromatography were the routine first level analysis for patients referred to our Hospital Center in Palermo to undergo the screening test for haemoglobinopathies. Sequencing analysis was the selected method for the phenotypic characterization, especially in case of new or very rare mutations in globin genes., Results: We report data of a rare single nucleotide variation at position -56 relative to transcription initiation site (NM_000518.4(HBB):c.-106G > C), identified in ten patients of Italian origin during the screening programme of the 'Sicilian population'. It was found in simple heterozygosity (n = 8), in association with beta haemoglobin variant Hb S (n = 1) and in heterozygosity with beta-thalassaemic allele IVS-I-1 G->A [(HBB):c.92 + 1G > A] and ααα anti3.7 rearrangement (n = 1)., Discussion: Heterozygous subjects for this substitution showed normal haematological and electrophoretic features. Heterozygotes for this mutation and other defect in globin genes showed the classical phenotype of a healthy carrier, therefore it can be considered a benign variant that does not alter the production and function of haemoglobin., Conclusion: This is another example of rare or new nucleotide variations whose identification and characterization is crucial in order to carry out appropriate genetic counselling to a potential risk couple.

Background: Thalassaemia and variant haemoglobin are the most common severe monogenic disorders worldwide., Aims: To develop prenatal diagnosis programmes for the prevention of the most important haemoglobin disorders and identify healthy carriers of thalassaemia., Methods: Sequencing analysis was used to obtain complete data on gene structure and to correlate specific phenotypic expression with mutations, especially for new or very rare mutations in globin genes., Results: A rare single nucleotide variation, HBB :c.93-23T>C, located in nucleotide 108 of the first intervening sequence of the HBB gene, was identified. This variation was previously reported but its clinical significance was not known. Six heterozygous patients had this nucleotide variation and eight further cases co-inherited it together with other defects in the globin genes. Heterozygous subjects for this substitution showed normal haematological and electrophoretic features, whereas subjects who were compound heterozygotes for this mutation and another defect in globin genes showed the classic phenotype of a healthy carrier., Conclusion: This nucleotide can be considered a single nucleotide polymorphism and not a thalassaemic mutation that reduces the production of haemoglobin. This is another example of a very rare nucleotide variation. Knowledge of this is important so that appropriate genetic counselling can be carried out of a couple potentially at risk, where one of the partners is a carrier of β-thalassaemia and the other is carrier of a nucleotide variation., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

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.