Search

Background Noonan and Noonan-like syndromes are multisystem genetic disorders, mainly with autosomal dominant trasmission, caused by mutations in several genes. Missense pathogenetic variants of SOS1 gene are the second most common cause of Noonan syndrome (NS) and account approximately for 13% to 17% of cases. Subjects carrying a pathogenetic variant in SOS1 gene tend to exhibit a distinctive phenotype that is characterized by ectodermal abnormalities. Cutis verticis gyrata (CVG) is a rare disease, congenital or acquired, characterized by the redundancy of skin on scalp, forming thick skin folds and grooves of similar aspect to cerebral cortex gyri. Several references in the literature have reported association between nonessential primary form of CVG and NS. Case presentation we report two cases of newborns with CVG and phenotype suggestive for NS who have been diagnosed to harbour the same pathogenetic variant in SOS1 gene. Conclusions previously described patients with NS presenting CVG had received only clinical diagnosis. Therefore we report the first patients with CVG in which the clinical suspicion of NS is confirmed by molecolar analysis.

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two β-globin gene defects, a β-thalassemia (β-thal), mutation a...

We report two very rare changes in the second intron of the HBB gene, a substitution at nucleotide (nt) 726 [IVS-II-726 (A>G) (β+), NM_000518, HBB: c.316-125A>G] and a deletion of a cytosine at nt 809 [IVS-II-809 (-C) (β), NM_000518, HBB: c.316-42delC] identified during the screening program for hemoglobinopathies in the resident Sicilian population. The purpose of this study was to evaluate the clinical implication of these rare changes, particularly in coinheritance with known mutations in the globin clusters, in order to conduct an appropriate genetic counseling for at-risk couples. Molecular analysis detected the first rare nt substitution in two cases in simple heterozygosity and in two cases in association with other known mutations on globin genes, while the deletion was identified in a pregnant woman, carrier of β-thal, and in her fetus at prenatal diagnosis (PND) for hemoglobinopathies. The present study emphasizes the importance of sharing the observed 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 and possible interactions with known molecular defects.

BackgroundThalassaemia and variant haemoglobin are the most common severe monogenic disorders worldwide.AimsTo develop prenatal diagnosis programmes for the prevention of the most important haemoglobin disorders and identify healthy carriers of thalassaemia.MethodsSequencing 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.ResultsA 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.ConclusionThis 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.

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two β-globin gene defects, a β-thalassemia (β-thal), mutation at IVS-I-110 (

Objective The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo-fetal cells. Method Celomic fluids were aspired by ultrasound-guided transcervical celocentesis at 7-9 weeks’ gestation from singleton pregnancies before surgical termination for psychological reasons. DNA was extracted from celomic fluid cells showed the same morphology and quantitative fluorescentPCR assay was performed to evaluate their fetal or maternal origin. Results Six different types of non-hematological maternal and four different types of embryo-fetal cells were detected. The most common maternal cells were of epithelial origin. The majority of embryo-fetal cells were roundish with a nucleus located in an eccentric position near the wall. These cells were considered to be erythroblasts, probably derived from the yolk sac that serves as the initial site of erythropoiesis. Conclusions The combined use of morphology and DNA analysis make it possible to select and isolate embryo-fetal cells, even when maternal contamination is high. This development provides the opportunity for the use of celocentesis for early prenatal diagnosis of genetic diseases and application of array-CGH. This article is protected by copyright. All rights reserved.

Objective Celocentesis, which involves aspiration of celomic fluid at 7–9 weeks' gestation, can potentially provide early prenatal diagnosis of single-gene disorders. The main barrier to wide acceptability of this technique is contamination of the sample by maternal cells. This problem can be overcome through selection of embryo-fetal erythroid precursors, which are found in celomatic fluid. Method Embryo-fetal erythroid precursors were selected by an anti-CD71 MicroBeads method or by direct micromanipulator pickup of the cells selected on the basis of their morphology. Results In our series of 302 singleton pregnancies at high risk for hemoglobinopathies, Celocentesis provided a sample of celomic fluid in all cases. In 100 (33.1%) samples, maternal contamination was absent or very low ( 60%, and selection of embryo-fetal cells was achieved by micromanipulation. In all 302 cases, there was concordance between DNA obtained from celomic fluid samples and fetal or newborn DNA. Conclusions Celocentesis can be a reliable procedure for earlier prenatal diagnosis of fetal monogenic diseases. © 2016 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 HbA2 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 HbA2 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 HbA2 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.

Coelomic fluid (CF) is the earliest dynamic and complex fluid of the gestational sac. CF contains maternal cells and proteins produced by embryonic cells, tissues and excretions. The biochemical composition of CF is modified throughout the first trimester of pregnancy and its protein profile reflects both physiological/pathological changes affecting the embryo and mother. Identification of variations in the balance of proteins might indicate particular types of pathologies, or ascertain specific genetic disorders. A platform utilizing protein enrichment procedures coupled with shotgun identification and iTRAQ differentiation provided the identification and quantitation of 88 unique embryonic proteins. It is relevant to note that chromosome X protein CXorf23 was found suggesting the embryo sex. Foetal sex was determined by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) on coelomic cells, foetal tissues and maternal white blood cells, with a 100% concordance rate between iTRAQ-MS/MS and QF-PCR data. The functional associations among the identified proteins were investigated using STRING database. Open Targets Platform showed as significant the following therapeutic areas: nervous, respiratory, eye and head system disease.

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.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.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 αααHeterozygous 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.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.

INTRODUCTION Currently, prenatal diagnosis of genetic disorders requires chorionic villus sampling or amniocentesis carried out after 11 and 16 weeks of gestation, respectively. Celocentesis is a procedure for prenatal diagnosis that could be used from as early as 7 weeks. The present investigation evaluated the feasibility of performing diagnosis for monogenic diseases using celomic fluid containing cells of fetal origin. MATERIAL AND METHODS Analysis consisted of 489 singleton pregnancies undergoing celocentesis for the prenatal diagnosis of hemoglobinopathies (n = 367) or before surgical termination of pregnancy for social indications (n = 122). Embryo-fetal cells were isolated from celomic fluid using CD71 antibodies or by micromanipulation. Quantitative fluorescent polymerase chain reaction of short tandem repeat sequences of chromosomes 13, 18, 21, X and Y were used to determine the presence of maternal DNA. RESULTS 357/489 (73%) of celomic fluid samples were contaminated with maternal cells. In two cases, diagnosis was not possible due to the high contamination of celomic fluid. Eighty-seven (23.8%) fetuses were affected by hemoglobinopathies and, in five cases, chromosomal aneuploidies were found, including three cases of trisomy 21, one of trisomy 13 and one of triploidy. In all cases, the diagnosis of hemoglobinopathies and chromosomal abnormalities was confirmed by molecular and traditional cytogenetic analysis after amniocentesis, chorionic villus or placental tissue collection following pregnancy termination. CONCLUSIONS The findings of this study demonstrate that embryo-fetal cell selection from celomic fluid allows reliable and early prenatal diagnosis of hemoglobinopathies and can give more information on any fetal aneuploidy following the control of maternal contamination by quantitative fluorescent-PCR.

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 αααanti3.7 arrangement, and the β hemoglobin variants Hb Gorwihl [β5(A2)Pro>Ala] and Hb City of Hope [β69(E13)Gly>Ser], found both in association with β0-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.

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.

The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo-fetal cells.Celomic fluids were aspired by ultrasound-guided transcervical celocentesis at 7-9 weeks' gestation from singleton pregnancies before surgical termination for psychological reasons. DNA extracted from celomic fluid cells showed the same morphology, and quantitative fluorescent polymerase chain reaction (PCR) assay was performed to evaluate their fetal or maternal origin.Six different types of non-hematological maternal and four different types of embryo-fetal cells were detected. The most common maternal cells were of epithelial origin. The majority of embryo-fetal cells were roundish with a nucleus located in an eccentric position near the wall. These cells were considered to be erythroblasts, probably derived from the yolk sac that serves as the initial site of erythropoiesis.The combined use of morphology and DNA analysis makes it possible to select and isolate embryo-fetal cells, even when maternal contamination is high. This development provides the opportunity for the use of celocentesis for early prenatal diagnosis of genetic diseases and application of array comparative genomic hybridization. © 2016 John WileySons, 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 (

Objectives: This report represents the first observation in Sicily of two rare b-globin gene variants, Hb Hershey [b70(E14) Ala fi Gly] and Hb La Pommeraie [b133(H11)Val fi Met], found in a 35-year-old male patient from Messina, in the north-east of Sicily during population screening for hemoglobinopathies. Methods: The occurrence of the Hb variants was assessed by cation exchange chromatography while complete blood counts were obtained using automatic cell counters. Red cell lysates were analyzed by electrophoresis at alkaline and acid pH. Stability of hemoglobin was checked by the isopropanol precipitation test and by the heat tests while inclusion bodies and reticulocyte count were determined by incubation of blood samples with brilliant cresyl blue. Molecular analysis was performed by DNA sequencing of b- and a-globin genes. Results: We observed an abnormally high performance liquid chromatography elution with a slight reduction in mean corpuscular volume and mean corpuscular haemoglobin parameters and mutations at codon 70 GCC fi GGC (Hb Hershey) and at codon 133 GTG fi ATG (Hb La Pommeraie) in b-globin gene. Conclusion: Family analysis of three generations demonstrated the presence of these two mutations in trans. So it was possible to describe the phenotypes of these variants in a heterozygous state and in double heterozygous state.

We report a retrospective analysis carried out on 23,485 subjects submitted to a screening program from 2000 to 2006. Of these subjects, 3,934 had borderline HbA(2) values from 3.1 to 3.9%; 410 samples, analyzed previously using PCR methods and sequencing because all of these were partners of a carrier of classical beta-thalassemia, were selected for statistical analysis. Of 410 subjects, 94 (22.9%) were positive for a molecular defect in the beta-, delta- or alpha-globin genes. The most prevalent molecular defects were beta IVS1 nt 6 (HBB c.92+6T C), co-inheritance of severe beta thalassemia and delta mutations, beta-promoter mutations and triplication of alpha genes were detected; alpha-thalassemia and Hb-variants were also evident. Borderline HbA(2) is not a rare event in a population with a high prevalence of beta-thalassemia carriers. These data support the necessity to investigate these cases at a molecular level, particularly if the partner is a carrier of beta-thalassemia.

A high oxygen affinity hemoglobin (Hb) variant, Hb J-Cape Town [alpha92(FG4)Arg-->Gln (alpha1), CGG-->CAG] was identified in a 30-year-old woman patient from Cosenza (Southern Italy) who had previously been diagnosed with juvenile polycythemia in other hospitals. The occurrence of the variant Hb was assessed by both cation exchange chromatography and liquid chromatography-mass spectrometry (LC-MS) analyses. A detailed structural and functional characterization of the variant was performed at both the protein and DNA level. Structural investigation of the Hb variant by mass spectrometric methodologies and peptide sequencing identified the amino acid replacement as Arg-->Gln at alpha92. The corresponding DNA mutation CGGCAG was assigned to codon 92 of the alpha1 gene by DNA sequencing. These findings highlight the importance of investigating the hypothesis of a high affinity variant in the presence of a polycythemia so as to avoid unnecessary bone marrow examination or radioactive treatment. This report represents the first observation of the Hb J-Cape Town variant in Italy.

Celocentesis, which involves aspiration of celomic fluid at 7-9 weeks' gestation, can potentially provide early prenatal diagnosis of single-gene disorders. The main barrier to wide acceptability of this technique is contamination of the sample by maternal cells. This problem can be overcome through selection of embryo-fetal erythroid precursors, which are found in celomatic fluid.Embryo-fetal erythroid precursors were selected by an anti-CD71 MicroBeads method or by direct micromanipulator pickup of the cells selected on the basis of their morphology.In our series of 302 singleton pregnancies at high risk for hemoglobinopathies, Celocentesis provided a sample of celomic fluid in all cases. In 100 (33.1%) samples, maternal contamination was absent or very low (5%), and unambiguous results were obtained without the need for any preliminary procedures. In 160 (53%) cases, the contamination was between 5% and 60%, and selection of embryo-fetal erythroid precursors was successfully achieved by anti-CD71 MicroBeads. In 42 (13.9%) cases, the contamination was60%, and selection of embryo-fetal cells was achieved by micromanipulation. In all 302 cases, there was concordance between DNA obtained from celomic fluid samples and fetal or newborn DNA.Celocentesis can be a reliable procedure for earlier prenatal diagnosis of fetal monogenic diseases.

Summary Introduction During an intensive screening program aimed at identifying the healthy carriers of thalassemia and the couples at risk of bearing an affected fetus, a rare single nucleotide variation (SNV), CAP + 1570 T > C (HBB:c*96T > C), located 12 nucleotides upstream of the polyadenylation signal in 3'UTR of the beta globin gene was identified. It was previously reported as a β+ thalassemia mutation and later as a plain polymorphism. Methods Genotype identification of globin gene mutations was carried out using sequencing analysis, GAP-PCR, and MLPA methods. Results CAP + 1570 T > C (HBB:c*96T > C) was found in 39 heterozygotes, in one case in homozygous state and in thirteen cases of co-inheritance of this nucleotide substitution with other mutations in globin genes. Carriers of this mutation showed a ‘silent’ phenotype without appreciable microcytosis and hypochromia, so they cannot be differentiated from noncarrier individuals. Compound heterozygotes for this mutation and severe β-thal mutations showed a variable phenotype ranging from β-thal carrier to mild form of β-thalassemia intermedia, revealing new aspects and allowing to better understand the clinical implications of this nucleotide substitution that can be classified as a silent β-thalassemic defect. Conclusion Data reported in this study indicate the need of investigating partner of β-thalassemia carrier by complete sequencing analysis of β-globin gene and of providing an appropriate genetic counseling for couples at risk undergoing prenatal diagnosis.

Summary Background Haemoglobinopathies are a major public health problem in Sicily: it was estimated a frequency of 1/245 couples are at risk of haemoglobinopathies. This paper reviews legislative actions, prevention activities, carrier screening, genetic counselling, foetal sampling and laboratory methodology analysis evolution reporting the results of 30 years of prevention actions to assess the efficiency of our preventative programme in the control of haemoglobinopathies in Sicily. Methods This programme consisted principally of five phases: legislative actions, public awareness campaign, carrier screening, genetic counselling and prenatal diagnosis. Results These programmes have been very effective, which we can see from a greater public awareness of thalassaemia and its prevention in the target population furthermore by a marked decline in the incidence of thalassaemia major and sickle cell anaemia from 1 in 245 live births in the absence of prevention to 1 in 2000, with a reduction in about 85%. The residual cases were because of a conscious choice by expecting parents in relation to improved life expectancy as well as improved quality of life of the affected patients. Conclusion The study suggests that public health authorities should act and invest in a similar programme for prevention of thalassaemia, as well as in relation to the increased survival of patients and the consequent organ complications.

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.

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.

Background and objective The inherited hemoglobinopathies are a large group of disorders that include thalassemias and hemoglobin variants. Accurate determination of the carrier phenotype is essential for detecting couples at risk for producing offspring with hemoglobinopathy. Heterozygous β-thalassemia is usually silent at the clinical level. His phenotype is characterized by microcytosis and hypochromia with increased hemoglobin A 2 (HbA 2 ) value. Therefore, HbA 2 determination plays a key role in screening programs for hemoglobinopathy. The aim of this review is to address and suggest an approach for reducing or abolishing hemoglobinopathy screening mistakes. Design and methods Quantitative methods for HbA 2 value determination, comment on the accuracy of the test and on the interpretation of data were discussed. The most probable diagnostic conclusion based on the HbA 2 level, hemoglobin pattern, hematological parameters and iron markers was suggested in this review. Results Hemoglobinopathies are the only genetic disease where it is possible to detect carriers using hematological findings rather than DNA analysis. However, hematological diagnosis is sometimes presumptive, and in these cases, DNA analysis becomes necessary. Complete screening is based on the detection of red cell indices, HbA 2 , HbF and hemoglobin variant values. In particular, HbA 2 determination plays a key role in screening programs for β-thalassemia because a small increase in this fraction is one of the most important markers of β-thalassemia heterozygous carriers. Conclusion Genetic factors both related and unrelated to the β- and α-globin gene clusters, iron metabolism, endocrinological disorders, and some types of anemia, together with intra- and inter-laboratory variations in HbA 2 determination, may cause difficulties in evaluating this measurement in screening programs for hemoglobinopathies. Therefore, knowledge of all these issues is important for reducing or eliminating the risk of mistakes in screening programs for hemoglobinopathies.

The increase of HbA2 is the most important feature in the identification of beta-thalassemia carriers. However, some carriers are difficult to identify, because the level of HbA2 is not in the typical range. Few data are available concerning the prevalence of such unusual phenotypes, and knowing their expected prevalence could be helpful in detecting systematic drifts in the analytical systems for HbA2 quantification. In this study we report a retrospective investigation in two centres with high prevalence of beta-thalassemia. The prevalence of borderline subjects was found to be 2.2 and 3.0%, respectively. The genotypes of a subgroup of these subjects were then analyzed and in about 25% of cases a mutation in the globin genes was identified. We conclude that the occurrence of HbA2 borderline phenotypes is not a rare event. In order to obtain more accurate HbA2 measurements the development of an international reference measurement system for HbA2, based on quantitative peptide mapping, has been recently started. We believe that the innovative approach of our method could also be used as a model to develop accurate quantitative methods for other red cell proteins relevant to the biodynamic properties and the surface electrochemistry of erythrocytes.

In countries with a high prevalence of hemoglobinopathy carriers, the only realistic approach to control the birth of new patients with thalassemia major or sickle cell disease is population screening in combination with invasive prenatal diagnosis[1]. In the early 1990's, molecular definition of the thalassemia defects, development of procedures for their detection by DNA analysis and introduction of amniotic fluid sampling (amniocentesis) or chorionic villus sampling (CVS) led to early prenatal diagnosis at 16 and 11 weeks, respectively [2]. An alternative technique for earlier diagnosis is celocentesis [3]. At the end of week 4 of gestation, the developing exocoelomic cavity (ECC) splits the extra-embryonic mesoderm into two layers, the somatic mesoderm lining the trophoblast and the splanchnic mesoderm covering the secondary yolk sac and the embryo. The ECC is the largest anatomical space inside the gestational sac between 5 and 9 weeks of gestation and is surrounded by celomic fluid (CF), which contains cells of fetal origin [4-5]. This fluid can be sampled by a technique that involves the ultrasound-guided insertion of a needle through the vagina from as early as 7 weeks of gestation. Previous studies utilizing coelocentesis for prenatal determination of single-gene defects reported variable success ranging from 58 to 95% because of presence of maternal cell contamination (MCC) [6]. In this work we demostred as this problem can be overcome through the identification of embryo-fetal erythroid precursors presented in the coelomatic fluid and their specific selection. 302 couples from different regions of Italy, at risk for ß thalassemia or sickle cell disease asked for prenatal diagnosis by coelocentesis that was carried out at between 6+6 and 9+2 weeks' gestation. Coelomic fluid samples with no or very low (5% maternal contamination, two different procedures were used to isolate embryo-fetal cells: the first technique involved positive selection of embryo-fetal erythroid precursors by anti-CD71 MicroBeads (160 samples), the second procedure was through the use of a micromanipulator (42 samples). In 68/300 (22.6%) cases the fetus was affected by ß-thalass/ß-thalassemia and 66 women chosen to terminate the pregnancy. Two families decided to continue the pregnancy for ethical reasons despite the documented presence of an affected fetus. The antenatal diagnosis was confirmed in all 66 cases by molecular analysis of placental tissue after termination (Table 1). In 232/300 (77.4%) cases the fetus was diagnosed as being normal or a carrier for ß-thalassemia or sicke cell disease. The results obtained after coelocentesis were confirmed by amniocentesis or postnatally. In two case (0.66%) was no obtained a reliable diagnosis because no fetal cells were found (Table 1). The findings of this study in a large number of pregnancies investigated by coelocentesis, demonstrate that embryo-fetal erytroid cell selection from coelomatic fluid allows reliable and earlier prenatal diagnosis of hemoglobinopathies. This technique is attractive to parents because it provides prenatal diagnosis of genetic disease at least 4 weeks earlier respect to traditional procedures reducing anxiety of parents and from a clinical point of view this procedure would allow women to undergo medical TOP at 8-10 weeks of gestation which is less traumatic and safer than second trimester surgical TOP. Table 1. Overall results of prenatal diagnosis of hemoglobinopathies by coelocentesis. Number of coelocentesis effectuated 302 Results of Prenatal diagnosis: Affected 68 Carrier of hemoglobinopathies 161 Non Affected 71 diagnosis not reiable 2 Control post coelocentesis: Placental tissue after termination 66 CVS or amniocentesis 153 Postnatal test 61 Awaiting 20 Coelocentesis diagnostic errors 0 Disclosures No relevant conflicts of interest to declare.

Although delta-globin gene (HBD MIM#142000) mutations have no clinical implications, co-inheritance of beta- and delta-thalassemia may lead to misdiagnosis. Among 7,153 samples studied for beta-thalassemia, 205 samples with lower than expected HbA2 levels were selected for our analysis and 183 samples (2.5%) were positive for delta-globin gene mutations. Twelve different mutations were detected, and among these five have not been not previously described (HbA2-Catania HBD c.8A--T, HbA2-Corleone HBD c.41C--A, HbA2-Ventimiglia HBD c.212C--G, HbA2-Montechiaro HBD c.260C--A, and HbA2-Bagheria HBD c.422C--T). This study suggests that delta-globin gene defects are very common in Sicily. Thus, these mutations need to be considered during beta-thalassemia screening to avoid false negative results in the detection of at-risk couples.

Hemoglobinopathies are the only genetic disease in which it is possible to detect carriers using hematologic findings rather than DNA analysis. Complete screening is based on the detection of red cell indices, HbA2, HbF and hemoglobin variant values. The classical phenotype of heterozygous β

The detection of β-thalassemia carriers is based on the correct estimation of HbA2 values.The diagnosis of β-thalassemia carriers may be more complicated in presence of δ-thalassemia because the interaction between δ and β-globin gene defects modifies the HbA2 values. For this reason, we studied the spectrum of δ-globin gene mutations present in the Sicilian population, characterized by its very high heterogeneity in β-thalassemia genotype and phenotype. The samples were selected by screening and routine prenatal diagnoses counseling for thalassemia. Among 7153 samples studied for β-thalassemia, 205 samples were selected for HbA2 levels ranging from 0.5% to 2.0% and normal haematology parameters, suspected of being δ-thal carriers in the absence of α and β-thalassemia or hemoglobin variants, and for HbA2 levels from 2.1% to 4.6% in subjects suspected of compound heterozygotes for δ and β-thalassemia and between 1.4% and 2.0% for δ and α-thalassemia. We have considered the value of 2% as the treshold between normal and d-carrier subjects. One-hundred-eighty-three samples showed to be positive for δ-globin gene mutations.Twelve mutations were detected and among these five were new δ-globin gene defects (HbA2-Catania, HbA2-Corleone, HbA2-Ventimiglia; HbA2-Montechiaro and HbA2-Bagheria) determining δ-globin gene variants and seven were already described mutations. Among these six of them were point mutations (HbA2-Mitsero, HbA2-NYU,HbA2-Yialousa, HbA2-Coburg, HbA2-Fitzroy) and one a 7.2 Kb deletion mutation known as the δ-Corfù type, HBD g.5946_1262del. As it was previously shown in Sicily for β-thalassemia, only three mutations account for 91% (HbA2-Yialousa, HbA2-NYU, IVS II-3′ A→G) of the overall δ-globin gene defects. HbA2-Yialousa (g.82G→T) is the most common mutation found in Sicilian population (81%) while the other eleven mutations are less frequent between 0.5 to 5.5%. These findings suggest that in Sicily δ-thalassemia is very common (2.5%) and as it was described, previously, for the β-thalassemia mutations,this also is very heterogeneous (twelve mutations). This information is noteworthy considering the implication that the interaction between β and δ-thalassemia could determine in terms of HbA2 decrease in subjects heterozygotes for β-thalassemia. HbA2 levels in δ-thalassemia and δ globin gene variants with and without interaction with α or β-globin gene mutations. The δ-alleles are all in heterozygote state except three cases with homozygosis. βA/βA αα/αα Gγ-158/Gγ 1.1 1 βA/βA αα/αα 1.7±0.2 113 βA/βA αα/αα 2.1±0.1 4 βA/βA αα/αα 0.7±0.1 3 βA/β(IVS1,110) αα/αα 3.35±0.15 2 βA/β(cd39) αα/αα 3.5 1 Hb A2-Yialousa βA/β(IVS1,1) αα/αα 3.5 1 βA/β(−87G) αα/αα 4.6 1 βA/β(−101) αα/αα 2.8 1 βA/β(IVS1,6) αα/αα 3.0±0.2 4 βA/βS αα/αα 2.7±0.1 5 βA/βA α−3.7α/αα 1.8±0.2 5 βA/βA α−20.5α/αα 1.4±0.1 2 βA/βA α−Medα/αα 1.6±0.1 2 βA/βA αNcoIα/αα 1.7±0.2 2 βA/βA αHphIα/αα 1.7±0.2 2 Hb A2-NYU βA/βA αα/αα 1.5±0.2 9 βA/β(Valletta) α−3.7α/αα 1.6 1 Hb A2-Mitsero βA/βA αα/αα 1.9±0.2 3 βA/βA α−3.7α/αα 1.6 1 Hb A2-Coburg βA/βA αα/αα 1 1 Hb A2-Fitzroy βA/βA αα/αα 1.4±0.2 3 Hb A2-Catania βA/βA αα/αα 1.2 1 Hb A2-Corleone βA/βA αα/αα 1.6 1 Hb A2-Ventimiglia βA/βA αα/αα 2 1 Hb A2-Montechiaro βA/βA αα/αα 1.3 1 Hb A2-Bagheria βA/βA αα/αα 1.7 1 7.2 Kb deletion βA/βA αα/αα 1.4±0.1 3 βA/βA αα/αα 1.5±0.2 4 IVS II-3′ βA/β(IVS1,110) αα/αα 3.3±0.2 3 βA/β αα/ααα 2.5 1 GENOTYPE δ GENOTYPEβ GENOTYPEα GENOTYPEγ HbA2% N° OF δ CARRIERS