Cutis verticis gyrata and Noonan syndrome: report of two cases with pathogenetic variant in SOS1 gene.

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.

Double Heterozygosity for Hb Durham-N.C. (HBB: c.344T>C) [ß114(G16)Leu→Pro] and the IVS-I-110 (HBB: c.93-21G>A) Causing a Severe ß-Thalassemia Phenotype.

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two ß-globin gene defects, a ß-thalassemia (ß-thal), mutation at IVS-I-110 (HBB: c.93-21G>A) and a variant hemoglobin (Hb) mutation at codon 114 (HBB: c.344T>C) on the other allele, reported as Hb Durham-N.C. (also known as Hb Brescia) [ß114(G16)Leu→Pro] in the HbVar database. A very low Hb level (Hb 3.5 g/dL), microcytosis [mean corpuscular volume (MCV) 63.2 fL] and hypocromia [mean corpuscular Hb (MCH) 19.6 pg], increased red blood cell (RBC) distribution width (RDW) (36.0%), higher reticulocytes (6.2%), anisocytosis, poikilocytosis, hypocromia, basophilic stippling and inclusion body formation, were present in the affected subject. Analysis of other family components showed the presence of HBB: c.93-21G>A defect in the mother and in her brother, while Hb Durham-N.C. was absent in all other relatives, thus, this mutation has arisen as a de novo defect. This is the first case described as a severe thalassemic phenotype in a compound heterozygote carrier of this unstable Hb and a common ß-thalassemic allele. The important information gained from this case is that a rare dominant or recessive mutation may arise in every individual, even if this is a very rare event.

Human coelomic fluid investigation: A MS-based analytical approach to prenatal screening.

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.

Fetal aneuploidy diagnosed at celocentesis for early prenatal diagnosis of congenital hemoglobinopathies.

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.

Co-inheritance of HBB:c.-106G > C, a rare single nucleotide variation at position -56 relative to transcription initiation site, with other known mutations in the globin clusters.

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.

HBB: c.316-125A>G and HBB: c.316-42delC: Phenotypic Evaluations of Two Rare Changes in the Second Intron of the HBB Gene.

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) (ß+), NM_000518, HBB: c.316-125A>G] and a deletion of a cytosine at nt 809 [IVS-II-809 (-C) (ß), 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 ß-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.

Phenotypic Evaluation of a Novel Nucleotide Substitution (HBD: c.442T>C) on the δ-Globin Gene.

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 ß-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 ß-thalassemia (ß-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.

Phenotypic evaluations of HBB:c.93-23T>C, a nucleotide substitution in the IVS I nt 108 of ß-globin gene.

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 ß-thalassaemia and the other is carrier of a nucleotide variation.

Identification of embryo-fetal cells in celomic fluid using morphological and short-tandem repeats analysis.

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 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. 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 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 Wiley & Sons, Ltd.

Coinheritance of a Rare Nucleotide Substitution on the ß-Globin Gene and Other Known Mutations in the Globin Clusters: Management in Genetic Counseling.

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 ß-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 ß(0) or ß(+) mutation. The purpose of this study was to evaluate the clinical implication of this nt change, particularly when coinherited with severe ß-thalassemia (ß-thal), in order to be able to conduct appropriate genetic counseling. Genetic studies were performed on two subjects, one carried Hb S [ß6(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 ß 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 ß-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.

Hb San Cataldo [ß144(HC1)Lys→Thr; HBB: C.434A > C]: A New Hemoglobin Variant with Increased Affinity for Oxygen.

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 ß-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 ß-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 ß chain. We called this new variant Hb San Cataldo for the native city of proband.

Embryo-fetal erythroid cell selection from celomic fluid allows earlier prenatal diagnosis of hemoglobinopathies.

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 (< 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 was > 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.

Co-inheritance of the rare ß hemoglobin variants Hb Yaounde, Hb Görwihl and Hb City of Hope with other alterations in globin genes: impact in genetic counseling.

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 ß 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 ß hemoglobin variants with other alterations in globin genes: the ß hemoglobin variant Hb Yaounde [ß134(H12)Val>Ala], found for the first time in combination with ααα(anti3.7) arrangement, and the ß hemoglobin variants Hb Görwihl [ß5(A2)Pro>Ala] and Hb City of Hope [ß69(E13)Gly>Ser], found both in association with ß(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.

Identification of three new nucleotide substitutions in the ß-globin gene: laboratoristic approach and impact on genetic counselling for beta-thalassaemia.

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 ß-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 ß-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.

The genetic heterogeneity of ß-globin gene defects in Sicily reflects the historic population migrations of the island.

The aim of this study is to update the incidence and the distribution of the globin gene defects causing ß-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 ß-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 ß-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.

Co-inheritance of Hb Hershey [beta70(E14) Ala-->Gly] and Hb La Pommeraie [beta133(H11)Val-->Met] in a Sicilian subject.

OBJECTIVES: This report represents the first observation in Sicily of two rare beta-globin gene variants, Hb Hershey [beta70(E14) Ala-->Gly] and Hb La Pommeraie [beta133(H11)Val-->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 beta- and alpha-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-->GGC (Hb Hershey) and at codon 133 GTG-->ATG (Hb La Pommeraie) in beta-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.

The significance of the hemoglobin A(2) value in screening for hemoglobinopathies.

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 beta-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 beta-thalassemia because a small increase in this fraction is one of the most important markers of beta-thalassemia heterozygous carriers. CONCLUSION: Genetic factors both related and unrelated to the beta- and alpha-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.

Hb Southern Italy: coexistence of two missence mutations (the Hb Sun Prairie alpha2 130 Ala --> Pro and Hb Caserta alpha2 26 Ala --> Thr) in a single HBA2 gene.

This study describes a new molecular condition in the alpha(2)-globin gene (HBA2) found in six unrelated families from Southern Italy (Campania and Sicily). This new double mutant form of haemoglobin is called Hb Southern Italy and originated from the coexistence of two known mutations occurring in the same globin gene, HBA2 26 G-->A (Hb Caserta) and HBA2 130 G-->C (Hb Sun Prairie). Hb Sun Prairie was originally observed in Indian patients in either the homozygous state, with severe hemolytic anemia, and in the heterozygous state with microcytosis, or in asymptomatic cases as an alpha-thalassemia carrier phenotype. Hb Caserta was observed for the first time in a Casertian family (South Italy) that displayed a slowmigrating haemoglobin upon investigation. We report the clinical phenotype and molecular study of this new double mutant form of haemoglobin in heterozygous and homozygous subjects, as well as in association with alpha degrees delectional thalassemia.